BICYCLIC PEPTIDE LIGANDS SPECIFIC FOR EphA2

ABSTRACT

The present invention relates to polypeptides which are covalently bound to non-aromatic molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the invention describes peptides which are high affinity binders of the Eph receptor tyrosine kinase A2 (EphA2). The invention also includes drug conjugates comprising said peptides, conjugated to one or more effector and/or functional groups, to pharmaceutical compositions comprising said peptide ligands and drug conjugates and to the use of said peptide ligands and drug conjugates in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/771,186, filed Jun. 9, 2020, which is a U.S. national stage application under 35 U.S.C. § 371 of International Application No. PCT/GB2018/053678, filed Dec. 19, 2018, which claims priority to United Kingdom Application No. 1721259.8, filed Dec. 19, 2017, United Kingdom Application No. 1804102.0, filed Mar. 14, 2018, and United Kingdom Application No. 1818603.1, filed Nov. 14, 2018, the entire contents of each of which are hereby incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 8, 2023, is named 199837_SL.xml and is 143,360 bytes in size.

FIELD OF THE INVENTION

The present invention relates to polypeptides which are covalently bound to non-aromatic molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the invention describes peptides which are high affinity binders of the Eph receptor tyrosine kinase A2 (EphA2). The invention also includes drug conjugates comprising said peptides, conjugated to one or more effector and/or functional groups, to pharmaceutical compositions comprising said peptide ligands and drug conjugates and to the use of said peptide ligands and drug conjugates in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

BACKGROUND OF THE INVENTION

Cyclic peptides are able to bind with high affinity and target specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355 Å²) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 Å²; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8, (MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin.

Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example TATA (1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one, Heinis et al. Angew Chem, Int Ed. 2014; 53:1602-1606).

Phage display-based combinatorial approaches have been developed to generate and screen large libraries of bicyclic peptides to targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclised by covalently linking the cysteine side chains to a small molecule scaffold.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a peptide ligand specific for EphA2 comprising a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a non-aromatic molecular scaffold which forms covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

According to a further aspect of the invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effector and/or functional groups.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand or a drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided a peptide ligand or drug conjugate as defined herein for use in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Body weight changes after administering BCY6031 to female Balb/C nude mice bearing LU-01-0046 tumor. Data points represent group mean body weight.

FIG. 2 : Tumor volume trace after administering BCY6031 to female Balb/C nude mice bearing LU-01-0046 tumor. Data points represent group mean. The treatment was ceased from day 28.

FIG. 3 : General schematic demonstrating the concept of preparing Bicycle drug conjugates (BDCs).

FIG. 4 : Plot of mean tumour volume versus time for BCY6136 in HT1080 xenograft mice. Doses (2, 3 and 5 mg/kg) were administered on days 0 and 7. Body weight changes during treatment indicative of tumour burden, drug-associated toxicology and overall animal health are illustrated in the top right inset.

FIG. 5 : Plot of mean tumour volume versus time for BCY6136 in NCI-H1975 xenograft mice. Doses (1, 2 and 3 mg/kg) were administered on days 0, 7, 14, 21, 28 and 35. Body weight changes during treatment indicative of tumour burden, drug-associated toxicology and overall animal health are illustrated in the top right inset.

FIG. 6 : Plot of mean tumour volume versus time for BCY6136 in MDA-MB-231 xenograft mice. Doses (1, 2 and 3 mg/kg) were administered on day 0, 7, 14, 21, 28, 35 and 45. Body weight changes during treatment indicative of tumour burden, drug-associated toxicology and overall animal health are illustrated in the top right inset.

FIGS. 7 to 9 : Body weight changes and tumor volume traces after administering BCY6136 (FIG. 7 ), ADC (FIG. 8 ) and BCY6033 (FIG. 9 ) to female BALB/c nude mice bearing PC-3 xenograft. Data points represent group mean body weight.

FIG. 10 : Body weight changes and tumor volume traces after administering BCY6136, EphA2-ADC or Docetaxel to male Balb/c nude mice bearing PC-3 xenograft. Data points represent group mean body weight.

FIGS. 11 to 13 : Body weight changes and tumor volume trace after administering BCY6033 (FIG. 11 ), BCY6136 (FIG. 12 ) and BCY6082 (FIG. 13 ) to female Balb/c nude mice bearing NCI-H1975 xenograft. Data points represent group mean tumor volume and body weight.

FIGS. 14 and 15 : Body weight changes and tumor volume traces after administering BCY6136 and ADC to female Balb/c nude mice bearing LU-01-0251 xenograft. Data points represent group mean body weight.

FIG. 16 : Body weight changes and tumor volume traces after administering BCY6033, BCY6136, BCY6082 and BCY6031 to female Balb/c nude mice bearing LU-01-0046. Data points represent group mean body weight.

FIG. 17 : Body weight changes and tumor volume traces after administering BCY6136 or ADC to female Balb/c nude mice bearing LU-01-0046 NSCLC PDX model. Data points represent group mean body weight.

FIGS. 18 to 22 : Body weight changes and tumor volume traces after administering BCY6033 (FIG. 18 ), BCY6136 (FIG. 19 ), BCY6082 (FIG. 20 ), BCY6173 (FIG. 21 ) and BCYs 6175 and 6031 (FIG. 22 ) to female Balb/c nude mice bearing LU-01-0046. Data points represent group mean body weight.

FIG. 23 : Body weight changes and tumor volume traces after administering BCY6136 (referred to in FIG. 23 as BT5528), BCY8245 or BCY8781 to female BALB/c nude mice bearing LU-01-0412 xenograft. Data points represent group mean tumor volume (left panel) and body weight (right panel).

FIG. 24 : Body weight changes and tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing LU-01-0486 xenograft. Data points represent group mean body weight.

FIGS. 25 to 27 : Body weight changes and tumor volume trace after administering BCY6033 (FIG. 25 ), BCY6136 (FIG. 26 ) and BCY6082 (FIG. 27 ) to female Balb/c nude mice bearing MDA-MB-231-luc xenograft. Data points represent group mean tumor volume and body weight.

FIG. 28 : Body weight changes and tumor volume traces after administering BCY6136 to female BALB/c mice bearing EMT-6 syngeneic. Data points represent group mean body weight. The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from Day 14.

FIG. 29 : Body weight changes and tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing NCI-N87 xenograft. Data points represent group mean body weight.

FIG. 30 : Body weight changes and tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing SK-OV-3 xenograft. Data points represent group mean body weight.

FIG. 31 : Body weight changes and tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing OE21 xenograft. Data points represent group mean body weight.

FIG. 32 : Body weight changes and tumor volume traces after administering BCY6136 to female CB17-SCID mice bearing MOLP-8 xenograft. Data points represent group mean body weight.

FIG. 33 : Body weight changes and Tumor volume traces after administering BCY6082 to female CB17-SCID mice bearing MOLP-8 xenograft. Data points represent group mean body weight.

FIGS. 34 to 42 : Body weight changes and tumor volume traces after administering BCY6082 (FIG. 34 , BCY6031 (FIG. 35 ), BCY6173 (FIG. 36 ), BCY6135 (FIG. 37 ), BCY6033 (FIG. 38 ), BCY6136 (FIG. 39 ), BCY6174 (FIG. 40 ), BCY6175 (FIG. 41 ) and ADC (FIG. 42 ) to female BALB/c nude mice bearing HT1080 xenograft. Data points represent group mean body weight.

Where error bars are present in the above Figures, these represent standard error of the mean (SEM).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, said loop sequences comprise 2, 3, 5, 6 or 7 amino acid acids.

In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 2 amino acids and the other of which consists of 7 amino acids (such as those listed in Table 4).

In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 5 amino acids (such as those listed in Tables 3 and 4).

In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids (such as those listed in Tables 3 to 5).

In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids (such as those listed in Table 10).

In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids (such as those listed in Table 4).

In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 7 amino acids (such as those listed in Table 5).

In one embodiment, the peptide ligand comprises an amino acid sequence selected from:

C_(i)-X₁-C_(ii)-X₂-C_(iii)

wherein X₁ and X₂ represent the amino acid residues between the cysteine residues listed in Tables 3 to 5 and C_(i), C_(ii) and C_(iii) represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.

In a further embodiment, the peptide ligand comprises an amino acid sequence selected from one or more of the peptide ligands listed in one or more Tables 3 to 5.

In a further embodiment, the peptide ligand comprises an amino acid sequence selected from:

C_(i)-X₁-C_(ii)-X₂-C_(iii)

wherein X₁ and X₂ represent the amino acid residues between the cysteine residues listed in Table 10 and C_(i), C_(ii) and C_(iii) represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.

In a further embodiment, the peptide ligand comprises an amino acid sequence selected from one or more of the peptide ligands listed in Table 10.

In one embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids and the peptide ligand has an amino acid sequence selected from:

(SEQ ID NO: 1) C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp)W(HArg)C_(iii); and (SEQ ID NO: 97) C_(i)PLVNPLC_(ii)LHPGWTCH_(iii); wherein HyP is hydroxyproline, HArg is homoarginine and C_(i), C_(ii) and C_(iii) represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.

In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids and the peptide ligand has the following amino acid sequence:

(SEQ ID NO: 1) C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp)W(HArg)C_(iii); wherein HyP is hydroxyproline, HArg is homoarginine and C_(i), C_(ii) and C_(iii) represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.

In one embodiment, the peptide ligand of the invention is a peptide ligand which is other than the amino acid sequence:

(SEQ ID NO: 1) C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp)W(HArg)C_(iii).

In one embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids and the peptide ligand has an amino acid sequence selected from:

(β-Ala)-Sar₁₀-A(HArg)D-C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp) W(HArg)C_(iii )(SEQ ID NO: 2) (BCY6099; Compound 66); and (β-Ala)-Sar₁₀-A(HArg)D-C_(i)PLVNPLC_(ii)LHPGWTC_(iii) ((β-Ala)-Sar₁₀-(SEQ ID NO: 11)) (BCY6014; Compound 67); wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.

In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids and the peptide ligand has the following amino acid sequence:

(β-Ala)-Sar₁₀-A(HArg)D-C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp) W(HArg)C_(iii )(SEQID NO: 2)(BCY6099; Compound 66); wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.

In one embodiment, the peptide ligand of the invention is a peptide ligand which is other than the amino acid sequence:

(β-Ala)-Sar₁₀-A(HArg)D-C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp) W(HArg)C_(iii )(SEQ ID NO: 2)(BCY6099; Compound 66).

In one embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and the peptide ligand is selected from any one of the peptide ligands listed in Tables 3 to 5.

In an alternative embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and the peptide ligand is selected from any one of the peptide ligands listed in Table 10.

In one embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and the peptide ligand is selected from:

(β-Ala)-Sar₁₀-A(HArg)D-C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp) W(HArg)C_(iii )(SEQ ID NO: 2)(BCY6099; Compound 66); and (β-Ala)-Sar₁₀-A(HArg)D-C_(i)PLVNPLC_(ii)LHPGWTC_(iii) ((β-Ala)-Sar₁₀-(SEQ ID NO: 11)) (BCY6014; Compound 67); wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.

In a further embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and the peptide ligand is:

(β-Ala)-Sar₁₀-A(HArg)D-C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp) W(HArg)C_(iii )(SEQ ID NO: 2); wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.

In one embodiment, the peptide ligand is selected from any one of Compounds 1-113 or a pharmaceutically acceptable salt thereof.

In a further embodiment, the peptide ligand is Compound 66 (BCY6099) or Compound 67 (BCY6014) or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, the peptide ligand is Compound 66 (BCY6099) or a pharmaceutically acceptable salt thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th) ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature

Numbering

When referring to amino acid residue positions within the peptides of the invention, cysteine residues (C_(i), C_(ii) and C_(iii)) are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within the peptides of the invention is referred to as below:

-C_(i)-HyP₁-L₂-V₃-N₄-P₅-L₆-C_(ii)-L₇-H₈-P₉-(D-Asp)₁₀- W₁₁-(HArg)₁₂-C_(iii)- (SEQ ID NO: 1).

For the purpose of this description, all bicyclic peptides are assumed to be cyclised with 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) yielding a tri-substituted 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tripropan-1-one structure. Cyclisation with TATA occurs on C_(i), C_(ii), and C_(iii).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal (β-Ala)-Sar₁₀-Ala tail would be denoted as:

(β-Ala)-Sar₁₀-A-(SEQ ID NO: X).

Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein would also find utility in their retro-inverso form. For example, the sequence is reversed (i.e. N-terminus become C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa).

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide, peptidic or peptidomimetic covalently bound to a molecular scaffold. Typically, such peptides, peptidics or peptidomimetics comprise a peptide having natural or non-natural amino acids, two or more reactive groups (i.e. cysteine residues) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide, peptidic or peptidomimetic is bound to the scaffold. In the present case, the peptides, peptidics or peptidomimetics comprise at least three cysteine residues (referred to herein as C_(i), C_(ii) and C_(iii)), and form at least two loops on the scaffold.

Advantages of the Peptide Ligands

Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:

-   -   Species cross-reactivity. This is a typical requirement for         preclinical pharmacodynamics and pharmacokinetic evaluation;     -   Protease stability. Bicyclic peptide ligands should in most         circumstances demonstrate stability to plasma proteases,         epithelial (“membrane-anchored”) proteases, gastric and         intestinal proteases, lung surface proteases, intracellular         proteases and the like. Protease stability should be maintained         between different species such that a bicyclic peptide lead         candidate can be developed in animal models as well as         administered with confidence to humans;     -   Desirable solubility profile. This is a function of the         proportion of charged and hydrophilic versus hydrophobic         residues and intra/inter-molecular H-bonding, which is important         for formulation and absorption purposes;     -   An optimal plasma half-life in the circulation. Depending upon         the clinical indication and treatment regimen, it may be         required to develop a bicyclic peptide with short or prolonged         in vivo exposure times for the management of either chronic or         acute disease states. The optimal exposure time will be governed         by the requirement for sustained exposure (for maximal         therapeutic efficiency) versus the requirement for short         exposure times to minimise toxicological effects arising from         sustained exposure to the agent;     -   Selectivity. Certain peptide ligands of the invention         demonstrate good selectivity over other Eph receptor tyrosine         kinases, such as EphA1, EphA3, EphA4, EphA5, EphA6, EphA7 and         EphB1 and factor XIIA, carbonic anhydrase 9 and CD38         (selectivity data for selected peptide ligands of the invention         may be seen in Tables 7 and 14). It should also be noted that         selected peptide ligands of the invention exhibit cross         reactivity with other species (eg mouse and rat) to permit         testing in animal models (Tables 3 to 6 and 15); and     -   Safety. Bleeding events have been reported in pre-clinical in         vivo models and clinical trials with EphA2 Antibody Drug         Conjugates. For example, a phase 1, open-label study with         MEDI-547 was halted due to bleeding and coagulation events that         occurred in 5 of 6 patients (Annunziata et al, Invest New         Drugs (2013) 31:77-84). The bleeding events observed in patients         were consistent with effects on the coagulation system observed         in rat and monkey pre-clinical studies: increased activated         partial thromboplastin time and increased fibrinogen/fibrin         degradation product (Annunziata et al IBID). Overt bleeding         events were reportedly seen in toxicology studies in monkeys         (Annunziata et al, IBID). Taken together these results imply         that MEDI-547 causes Disseminated Intravascular Coagulation         (DIC) in both preclinical species and patients. The BDCs         reported here have short in vivo half lives (<30 minutes) and         are therefore intrinsically less likely to give rise to DIC in         patients. Results shown here (see BIOLOGICAL DATA sections 5 and         6 and Table 20) demonstrate that selected Bicycle Drug         Conjugates of the invention have no effect on coagulation         parameters and gave rise to no bleeding events in pre-clinical         studies.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.

The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may be anionic (e.g., —COOH may be —COO⁻), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

Where the peptides of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with one or more replacement amino acids, such as an alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol-reactive reagents so as to functionalise said amino acids, and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively.

In one embodiment, the modified derivative comprises an N-terminal and/or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry, and/or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C-terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.

In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal residue is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide.

In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target.

In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal residue is synthesized as an amide during peptide synthesis leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected having isosteric/isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.

Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, CD-disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine (C_(i)) and/or the C-terminal cysteine (C_(iii)).

In one embodiment, the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues. In a further embodiment, the modified derivative comprises replacement of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resultant bicyclic peptide ligand.

In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

In one embodiment, the modified derivative comprises replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise i-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines, such as D-alanines. This embodiment provides the advantage of identifying key binding residues and removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:

-   -   Incorporating hydrophobic moieties that exploit the hydrophobic         effect and lead to lower off rates, such that higher affinities         are achieved;     -   Incorporating charged groups that exploit long-range ionic         interactions, leading to faster on rates and to higher         affinities (see for example Schreiber et al, Rapid,         electrostatically assisted association of proteins (1996),         Nature Struct. Biol. 3, 427-31); and     -   Incorporating additional constraint into the peptide, by for         example constraining side chains of amino acids correctly such         that loss in entropy is minimal upon target binding,         constraining the torsional angles of the backbone such that loss         in entropy is minimal upon target binding and introducing         additional cyclisations in the molecule for identical reasons.         (for reviews see Gentilucci et al, Curr. Pharmaceutical Design,         (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem         (2009), 16, 4399-418).

Isotopic Variations

The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.

Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T), carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³¹C, fluorine, such as ¹⁸F, iodine, such as ¹²³I, ¹²⁵I and ¹³¹I, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹¹O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur, such as ³S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga, yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as ²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies, and to clinically assess the presence and/or absence of the EphA2 target on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

Non-Aromatic Molecular Scaffold

References herein to the term “non-aromatic molecular scaffold” refer to any molecular scaffold as defined herein which does not contain an aromatic (i.e. unsaturated) carbocyclic or heterocyclic ring system.

Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

As noted in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule.

In one embodiment the molecular scaffold may be a macromolecule. In one embodiment the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

In one embodiment the molecular scaffold comprises reactive groups that are capable of reacting with functional group(s) of the polypeptide to form covalent bonds.

The molecular scaffold may comprise chemical groups which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

An example of an a unsaturated carbonyl containing compound is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

Effector and Functional Groups

According to a further aspect of the invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effector and/or functional groups.

Effector and/or functional groups can be attached, for example, to the N and/or C termini of the polypeptide, to an amino acid within the polypeptide, or to the molecular scaffold.

Appropriate effector groups include antibodies and parts or fragments thereof. For instance, an effector group can include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to the one or more constant region domains. An effector group may also comprise a hinge region of an antibody (such a region normally being found between the CH1 and CH2 domains of an IgG molecule).

In a further embodiment of this aspect of the invention, an effector group according to the present invention is an Fc region of an IgG molecule. Advantageously, a peptide ligand-effector group according to the present invention comprises or consists of a peptide ligand Fc fusion having a tβ half-life of a day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more or 7 days or more. Most advantageously, the peptide ligand according to the present invention comprises or consists of a peptide ligand Fc fusion having a tβ half-life of a day or more.

Functional groups include, in general, binding groups, drugs, reactive groups for the attachment of other entities, functional groups which aid uptake of the macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate into cells will allow peptides against intracellular targets to be effective. Targets that can be accessed by peptides with the ability to penetrate into cells include transcription factors, intracellular signalling molecules such as tyrosine kinases and molecules involved in the apoptotic pathway. Functional groups which enable the penetration of cells include peptides or chemical groups which have been added either to the peptide or the molecular scaffold. Peptides such as those derived from such as VP22, HIV-Tat, a homeobox protein of Drosophila (Antennapedia), e.g. as described in Chen and Harrison, Biochemical Society Transactions (2007) Volume 35, part 4, p 821; Gupta et al. in Advanced Drug Discovery Reviews (2004) Volume 57 9637. Examples of short peptides which have been shown to be efficient at translocation through plasma membranes include the 16 amino acid penetratin peptide from Drosophila Antennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p 10444), the 18 amino acid ‘model amphipathic peptide’ (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p 127) and arginine rich regions of the HIV TAT protein. Non peptidic approaches include the use of small molecule mimics or SMOCs that can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p 153). Other chemical strategies to add guanidinium groups to molecules also enhance cell penetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p 13585). Small molecular weight molecules such as steroids may be added to the molecular scaffold to enhance uptake into cells.

One class of functional groups which may be attached to peptide ligands includes antibodies and binding fragments thereof, such as Fab, Fv or single domain fragments. In particular, antibodies which bind to proteins capable of increasing the half-life of the peptide ligand in vivo may be used.

In one embodiment, a peptide ligand-effector group according to the invention has a tβ half-life selected from the group consisting of: 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more or 20 days or more. Advantageously a peptide ligand-effector group or composition according to the invention will have a tβ range 12 to 60 hours. In a further embodiment, it will have a tβ half-life of a day or more. In a further embodiment still, it will be in the range 12 to 26 hours.

In one particular embodiment of the invention, the functional group is selected from a metal chelator, which is suitable for complexing metal radioisotopes of medicinal relevance.

Possible effector groups also include enzymes, for instance such as carboxypeptidase G2 for use in enzyme/prodrug therapy, where the peptide ligand replaces antibodies in ADEPT.

In one particular embodiment of the invention, the functional group is selected from a drug, such as a cytotoxic agent for cancer therapy. Suitable examples include: alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; Anti-metabolites including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids such as Vincristine, Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin and its derivatives etoposide and teniposide; Taxanes, including paclitaxel, originally known as Taxol; topoisomerase inhibitors including camptothecins: irinotecan and topotecan, and type II inhibitors including amsacrine, etoposide, etoposide phosphate, and teniposide. Further agents can include antitumour antibiotics which include the immunosuppressant dactinomycin (which is used in kidney transplantations), doxorubicin, epirubicin, bleomycin, calicheamycins, and others.

In one further particular embodiment of the invention, the cytotoxic agent is selected from maytansinoids (such as DM1) or monomethyl auristatins (such as MMAE).

DM1 is a cytotoxic agent which is a thiol-containing derivative of maytansine and has the following structure:

Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent and has the following structure:

In one yet further particular embodiment of the invention, the cytotoxic agent is selected from maytansinoids (such as DM1). Data is presented herein in Table 6 which demonstrates the effects of peptide ligands conjugated to toxins containing DM1.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulphide bond or a protease sensitive bond. In a further embodiment, the groups adjacent to the disulphide bond are modified to control the hindrance of the disulphide bond, and by this the rate of cleavage and concomitant release of cytotoxic agent.

Published work established the potential for modifying the susceptibility of the disulphide bond to reduction by introducing steric hindrance on either side of the disulphide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction by intracellular glutathione and also extracellular (systemic) reducing agents, consequentially reducing the ease by which toxin is released, both inside and outside the cell. Thus, selection of the optimum in disulphide stability in the circulation (which minimises undesirable side effects of the toxin) versus efficient release in the intracellular milieu (which maximises the therapeutic effect) can be achieved by careful selection of the degree of hindrance on either side of the disulphide bond.

The hindrance on either side of the disulphide bond is modulated through introducing one or more methyl groups on either the targeting entity (here, the bicyclic peptide) or toxin side of the molecular construct.

In one embodiment, the drug conjugate additionally comprises a linker between said peptide ligand and said cytotoxic agents.

In one embodiment, the cytotoxic agent and linker is selected from any combinations of those described in WO 2016/067035 (the cytotoxic agents and linkers thereof are herein incorporated by reference).

In one embodiment the cytotoxic agent is selected from DM1 or MMAE.

In one embodiment, the linker between said cytotoxic agent and said bicyclic peptide comprises one or more amino acid residues. Thus, in one embodiment, the cytotoxic agent is MMAE and the linker is selected from: -Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-, -Ala-Ala-Asn-, D-Ala-Phe-Lys- or -Glu-Pro-Cit-Gly-hPhe-Tyr-Leu- (SEQ ID NO: 98). In a further embodiment, the cytotoxic agent is MMAE and the linker is selected from: -Val-Cit-, -Trp-Cit-, -Val-Lys- or -D-Trp-Cit-. In a yet further embodiment, the cytotoxic agent is MMAE and the linker is -Val-Cit- or -Val-Lys-. In a still yet further embodiment, the cytotoxic agent is MMAE and the linker is -Val-Cit-.

In an alternative embodiment, the linker between said cytoxic agent comprises a disulfide bond, such as a cleavable disulfide bond. Thus, in a further embodiment, the cytotoxic agent is DM1 and the linker is selected from: —S—S—, —SS(SO₃H)—, —SS-(Me)-, -(Me)-SS-(Me)-, —SS-(Me₂)- or —SS-(Me)-SO₃H—. In a further embodiment, the cytotoxic agent is DM1 and the linker comprises an —S—S— moiety, such as (N-succinimidyl 3-(2-pyridyldithio)propionate (SPDB), or an —SS(SO₃H)— moiety, such as SO₃H-SPDB.

In an alternative embodiment, the cytotoxic agent comprises a non-cleavable cytotoxic agent. Thus, in one embodiment the cytotoxic agent is non-cleavable MMAE (such as the cytotoxic agent within BCY6063) or non-cleavable DM1 (such as the cytotoxic agent within BCY6064).

In one embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (A):

wherein said bicycle is selected from any one of BCY6099 and BCY6014 as defined herein.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B):

wherein said bicycle is selected from any one of BCY6099 and BCY6014 as defined herein.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (A), wherein said bicycle is selected from BCY6099 as defined herein. This BDC is known herein as BCY6027. Data is presented herein which demonstrates excellent competition binding for BCY6027 in the EphA2 competition binding assay as shown in Table 6.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B), wherein said bicycle is selected from BCY6099 as defined herein. This BDC is known herein as BCY6028. Data is presented herein which demonstrates excellent competition binding for BCY6028 in the EphA2 competition binding assay as shown in Table 6.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (A), wherein said bicycle is selected from BCY6014 as defined herein. This BDC is known herein as BCY6031. Data is presented herein which demonstrates excellent competition binding for BCY6031 in the EphA2 competition binding assay as shown in Table 6. Data is also presented herein in Table 11 and FIGS. 1 and 2 which demonstrate that BCY6031 treatment completely eradicated non-small cell lung carcinomas from day 32 and no tumour regrowth occurred following dosing suspension on day 28.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B), wherein said bicycle is selected from BCY6014 as defined herein. This BDC is known herein as BCY6032. Data is presented herein which demonstrates excellent competition binding for BCY6032 in the EphA2 competition binding assay as shown in Table 6.

In an alternative embodiment, the cytotoxic agent is MMAE or DM1 and the drug conjugate is selected from any of the BDCs listed in Table 11. Data is presented herein which shows that these BDCs exhibited excellent cross reactivity between human, mouse and rodent EphA2 as shown in Table 11.

In a further embodiment, the cytotoxic agent is MMAE or DM1 and the drug conjugate is selected from any of the BDCs listed in Table 13.

In a further embodiment, the cytotoxic agent is MMAE or DM1 and the drug conjugate is selected from BCY6033, BCY6082, BCY6136 and BCY6173. Data is presented herein which shows that these four Bicycle Drug Conjugates exhibited no significant binding to: closely related human homologs EphA1, EphA3, EphA4, EphA5, EphA6, EphA7 and EphB4; mouse EphA3 and EphA4; and rat EphA3 and EphB1 as shown in Tables 14 and 15.

In a yet further embodiment, the drug conjugate is selected from any one of: BCY6031, BCY6033, BCY6082, BCY6135, BCY6136, BCY6173, BCY6174 and BCY6175:

In one embodiment, the drug conjugate is other than BCY6027, BCY6028, BCY6135, BCY6136, BCY6173, BCY6174 and BCY6175.

In a still yet further embodiment, the drug conjugate is BCY6136. Data is presented herein in Studies 7 and 8 which show that BCY6136 showed significant and potent anti-tumor activity in the PC-3 xenograft prostate cancer model (see FIGS. 7 to 10 and Tables 21 to 24). Data is also provided herein which show that BCY6136 demonstrated potent antitumor activity in the NCI-H1975 xenograft lung cancer (NSCLC) model (see FIGS. 11 to 13 and Tables 25 to 30). Data is also presented herein in Studies 10 and 11 which show that BCY6136 demonstrated potent anti-tumor effect in both large and small tumour size LU-01-0251 PDX lung cancer (NSCLC) models (see FIGS. 14 and 15 and Tables 31 to 34) wherein complete tumor regression was observed. Data is also presented herein in Study 12 which show that BCY6136 demonstrated significant anti-tumor effect in the LU-01-0046 PDX lung cancer (NSCLC) model (see FIG. 16 and Tables 35 and 36) wherein complete tumor regression was observed for BCY6136. Data is also presented herein in Study 13 which show that BCY6136 demonstrated dose dependent anti-tumor activity in the LU-01-0046 PDX lung cancer (NSCLC) model (see FIG. 17 and Tables 37 and 38). Data is also presented herein in Study 14 which show BCY6136 eradicated tumors in the LU-01-0046 PDX lung cancer (NSCLC) model (see FIGS. 18 to 22 and Tables 39 to 42). Data is also presented herein in Studies 15 and 16 which demonstrate the effects of BCY6136 in two models which make use of cell lines with low/negligible EphA2 expression (namely Lu-01-0412 and Lu-01-0486). This data is shown in FIGS. 23 and 24 and Tables 43 to 46 and demonstrate that BCY6136 had no effect upon tumor regression in either cell line but BCYs BCY8245 and BCY8781, which bind to a target highly expressed in the Lu-01-0412 cell line, completely eradicated the tumour. Data is presented herein in Study 17 which show that BCY6136 demonstrated potent antitumor activity in the MDA-MB-231 xenograft breast cancer model (see FIGS. 25 to 27 and Tables 47 to 50). Data is also presented herein in Study 18 which demonstrates the effects of BCY6136 in a breast cancer model which makes use of a cell line with low/negligible EphA2 expression (namely EMT6). This data is shown in FIG. 28 and Tables 51 and 52 and demonstrates that BCY6136 had no effect upon tumor regression in this cell line. Data is also presented herein in Study 19 which show that BCY6136 demonstrated significant antitumor activity in the NCI-N87 xenograft gastric cancer model (see FIG. 29 and Tables 53 and 54). Data is also presented herein in Study 20 which show that BCY6136 demonstrated significant antitumor activity in the SK-OV-3 xenograft ovarian cancer model (see FIG. 30 and Tables 55 and 56) compared with the ADC MEDI-547 which demonstrated moderate antitumour activity. Data is also presented herein in Study 21 which show that BCY6136 demonstrated significant antitumor activity in the OE-21 xenograft oesophageal cancer model (see FIG. 31 and Tables 57 and 58). Data is also presented herein in Study 22 which show that BCY6136 demonstrated dose-dependent antitumor activity in the MOLP-8 xenograft multiple myeloma model and BCY6082 demonstrated significant antitumor activity (see FIGS. 32 and 33 and Tables 59 and 60). Data is also presented herein in Study 23 which show that BCY6136 demonstrated potent antitumor activity in the HT-1080 xenograft fibrosarcoma model (see FIGS. 34 to 41 and Tables 61 and 62).

Synthesis

The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et al (supra).

Thus, the invention also relates to manufacture of polypeptides or conjugates selected as set out herein, wherein the manufacture comprises optional further steps as explained below. In one embodiment, these steps are carried out on the end product polypeptide/conjugate made by chemical synthesis.

Optionally amino acid residues in the polypeptide of interest may be substituted when manufacturing a conjugate or complex.

Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at its N-terminus or C-terminus or within the loops using orthogonally protected lysines (and analogues) using standard solid phase or solution phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus. Alternatively additions may be made by fragment condensation or native chemical ligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, for example using subtiligase as described in (Chang et al Proc Natl Acad Sci USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages 6000-6003).

Alternatively, the peptides may be extended or modified by further conjugation through disulphide bonds. This has the additional advantage of allowing the first and second peptide to dissociate from each other once within the reducing environment of the cell. In this case, the molecular scaffold could be added during the chemical synthesis of the first peptide so as to react with the three cysteine groups; a further cysteine or thiol could then be appended to the N or C-terminus of the first peptide, so that this cysteine or thiol only reacted with a free cysteine or thiol of the second peptide, forming a disulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.

Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand or a drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum and immunotoxins. Pharmaceutical compositions can include “cocktails” of various cytotoxic or other agents in conjunction with the protein ligands of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. Preferably, the pharmaceutical compositions according to the invention will be administered by inhalation. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter indications and other parameters to be taken into account by the clinician.

The peptide ligands of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that levels may have to be adjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a “therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.

A composition containing a peptide ligand according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the peptide ligands described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.

Therapeutic Uses

The bicyclic peptides of the invention have specific utility as EphA2 binding agents.

Eph receptor tyrosine kinases (Ephs) belong to a large group of receptor tyrosine kinases (RTKs), kinases that phosphorylate proteins on tyrosine residues. Ephs and their membrane bound ephrin ligands (ephrins) control cell positioning and tissue organization (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur at higher ligand oligomerization states (Stein et al. (1998) Genes Dev 12, 667-678).

Among other patterning functions, various Ephs and ephrins have been shown to play a role in vascular development. Knockout of EphB4 and ephrin-B2 results in a lack of the ability to remodel capillary beds into blood vessels (Poliakov et al., supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrins has also been observed in newly-formed, adult micro-vessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

The de-regulated re-emergence of some ephrins and their receptors in adults also has been observed to contribute to tumor invasion, metastasis and neo-angiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., supra). Furthermore, some Eph family members have been found to be over-expressed on tumor cells from a variety of human tumors (Brantley-Sieders et al., supra); Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humans is encoded by the EPHA2 gene.

EphA2 is upregulated in multiple cancers in man, often correlating with disease progression, metastasis and poor prognosis e.g.: breast (Zelinski et al (2001) Cancer Res. 61, 2301-2306; Zhuang et al (2010) Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS One 6, e24426), lung (Brannan et al (2009) Cancer Prev Res (Phila) 2, 1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo et al (2013) J Thorac Oncol. 8, 301-308), gastric (Nakamura et al (2005) Cancer Sci. 96, 42-47; Yuan et al (2009) Dig Dis Sci 54, 2410-2417), pancreatic (Mudali et al (2006) Clin Exp Metastasis 23, 357-365), prostate (Walker-Daniels et al (1999) Prostate 41, 275-280), liver (Yang et al (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky et al (2005) Mol Cancer Res. 3, 541-551; Li et al (2010) Tumour Biol. 31, 477-488).

The full role of EphA2 in cancer progression is still not defined although there is evidence for interaction at numerous stages of cancer progression including tumour cell growth, survival, invasion and angiogenesis. Downregulation of EphA2 expression suppresses tumour cancer cell propagation (Binda et al (2012) Cancer Cell 22, 765-780), whilst EphA2 blockade inhibits VEGF induced cell migration (Hess et al (2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng et al (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al (2005) FASEB J. 19, 1884-1886).

An antibody drug conjugate to EphA2 has been shown to significantly diminish tumour growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68, 9367-9374) and a similar approach has been tried in man although treatment had to be discontinued for treatment related adverse events (Annunziata et al (2013) Invest New drugs 31, 77-84).

Polypeptide ligands selected according to the method of the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like. Ligands having selected levels of specificity are useful in applications which involve testing in non-human animals, where cross-reactivity is desirable, or in diagnostic applications, where cross-reactivity with homologues or paralogues needs to be carefully controlled. In some applications, such as vaccine applications, the ability to elicit an immune response to predetermined ranges of antigens can be exploited to tailor a vaccine to specific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the selected polypeptides may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

According to a further aspect of the invention, there is provided a peptide ligand or a drug conjugate as defined herein, for use in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

According to a further aspect of the invention, there is provided a method of preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour), which comprises administering to a patient in need thereof an effector group and drug conjugate of the peptide ligand as defined herein.

In one embodiment, the EphA2 is mammalian EphA2. In a further embodiment, the mammalian EphA2 is human EphA2.

In one embodiment, the disease or disorder characterised by overexpression of EphA2 in diseased tissue is selected from cancer.

Examples of cancers (and their benign counterparts) which may be treated (or inhibited) include, but are not limited to tumours of epithelial origin (adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cell carcinomas and other carcinomas) such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gall bladder and biliary system, exocrine pancreas, kidney, lung (for example adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomas and mesotheliomas), head and neck (for example cancers of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands, nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (for example thyroid follicular carcinoma), adrenal, prostate, skin and adnexae (for example melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic naevus); haematological malignancies (i.e. leukemias, lymphomas) and premalignant haematological disorders and disorders of borderline malignancy including haematological malignancies and related conditions of lymphoid lineage (for example acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas, Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and haematological malignancies and related conditions of myeloid lineage (for example acute myelogenousleukemia [AML], chronic myelogenousleukemia [CML], chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythaemia vera, essential thrombocythaemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocyticleukemia); tumours of mesenchymal origin, for example sarcomas of soft tissue, bone or cartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumours, benign and malignant histiocytomas, and dermatofibrosarcomaprotuberans; tumours of the central or peripheral nervous system (for example astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumours and schwannomas); endocrine tumours (for example pituitary tumours, adrenal tumours, islet cell tumours, parathyroid tumours, carcinoid tumours and medullary carcinoma of the thyroid); ocular and adnexal tumours (for example retinoblastoma); germ cell and trophoblastic tumours (for example teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and paediatric and embryonal tumours (for example medulloblastoma, neuroblastoma, Wilms tumour, and primitive neuroectodermal tumours); or syndromes, congenital or otherwise, which leave the patient susceptible to malignancy (for example Xeroderma Pigmentosum).

In a further embodiment, the cancer is selected from: breast cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, liver cancer, glioblastoma and angiogenesis.

In a further embodiment, the cancer is selected from: prostate cancer, lung cancer (such as non-small cell lung carcinomas (NSCLC)), breast cancer (such as triple negative breast cancer), gastric cancer, ovarian cancer, oesophageal cancer, multiple myeloma and fibrosarcoma.

In a yet further embodiment, the cancer is prostate cancer. Data is presented herein in Studies 7 and 8 which show that BCY6033 and BCY6136 showed significant and potent anti-tumor activity in the PC-3 xenograft prostate cancer model (see FIGS. 7 to 10 and Tables 21 to 24).

In a yet further embodiment, the drug conjugate is useful for preventing, suppressing or treating solid tumours such as fibrosarcomas and breast, and non-small cell lung carcinomas.

In a yet further embodiment, the cancer is selected from lung cancer, such as non-small cell lung carcinomas (NSCLC). Data is presented herein which demonstrates that a BDC of the invention (BCY6031) completely eradicated non-small cell lung carcinomas from day 32 and no tumour regrowth occurred following dosing suspension on day 28. This data clearly demonstrates the clinical utility of the BDCs of the present invention in cancers such as lung cancers, in particular non-small cell lung carcinomas. Data is also presented herein in Study 9 which show that BCY6033 demonstrated dose dependent anti-tumor activity, BCY6082 demonstrated significant antitumor activity and BCY6136 demonstrated potent antitumor activity in the NCI-H1975 xenograft lung cancer (NSCLC) model (see FIGS. 11 to 13 and Tables 25 to 30). Data is also presented herein in Studies 10 and 11 which show that BCY6136 demonstrated potent anti-tumor effect in both large and small tumour size LU-01-0251 PDX lung cancer (NSCLC) models (see FIGS. 14 and 15 and Tables 31 to 34) wherein complete tumor regression was observed. Data is also presented herein in Study 12 which show that BCY6033, BCY6136, BCY6082 and BCY6031 demonstrated significant anti-tumor effect in the LU-01-0046 PDX lung cancer (NSCLC) model (see FIG. 16 and Tables 35 and 36) wherein complete tumor regression was observed for BCY6033 and BCY6136. Data is also presented herein in Study 13 which show that BCY6136 demonstrated dose dependent anti-tumor activity in the LU-01-0046 PDX lung cancer (NSCLC) model (see FIG. 17 and Tables 37 and 38). Data is also presented herein in Study 14 which show that BCY6082 demonstrated dose dependent antitumor activity, BCY6031 and BCY6173 demonstrated antitumor activity and BCY6033, BCY6136 and BCY6175 eradicated tumors in the LU-01-0046 PDX lung cancer (NSCLC) model (see FIGS. 18 to 22 and Tables 39 to 42). Data is also presented herein in Studies 15 and 16 which demonstrate the effects of BCY6136 in two models which make use of cell lines with low/negligible EphA2 expression (namely Lu-01-0412 and Lu-01-0486). This data is shown in FIGS. 23 and 24 and Tables 43 to 46 and demonstrate that BCY6136 had no effect upon tumor regression in either cell line but BCYs BCY8245 and BCY8781, which bind to a target highly expressed in the Lu-01-0412 cell line, completely eradicated the tumour. In a further embodiment, the cancer is breast cancer. In a yet further embodiment, the breast cancer is triple negative breast cancer. Data is presented herein in Study 17 which show that BCY6082 demonstrated anti-tumor activity, BCY6033 demonstrated dose dependent antitumor activity and BCY6136 demonstrated potent antitumor activity in the MDA-MB-231 xenograft breast cancer model (see FIGS. 25 to 27 and Tables 47 to 50). Data is also presented herein in Study 18 which demonstrates the effects of BCY6136 in a breast cancer model which makes use of a cell line with low/negligible EphA2 expression (namely EMT6). This data is shown in FIG. 28 and Tables 51 and 52 and demonstrates that BCY6136 had no effect upon tumor regression in this cell line. In an alternative embodiment, the breast cancer is Herceptin resistant breast cancer. Without being bound by theory, EphA2 is believed to be implicated in the resistance to Herceptin, therefore, an EphA2-targeting entity has potential utility in patients who have failed to respond to Herceptin.

In a further embodiment, the cancer is gastric cancer. Data is presented herein in Study 19 which show that BCY6136 demonstrated significant antitumor activity in the NCI-N87 xenograft gastric cancer model (see FIG. 29 and Tables 53 and 54).

In a further embodiment, the cancer is ovarian cancer. Data is presented herein in Study 20 which show that BCY6136 demonstrated significant antitumor activity in the SK-OV-3 xenograft ovarian cancer model (see FIG. 30 and Tables 55 and 56) compared with the ADC MEDI-547 which demonstrated moderate antitumour activity.

In a further embodiment, the cancer is oesophageal cancer. Data is presented herein in Study 21 which show that BCY6136 demonstrated significant antitumor activity in the OE-21 xenograft oesophageal cancer model (see FIG. 31 and Tables 57 and 58).

In a further embodiment, the cancer is multiple myeloma. Data is presented herein in Study 22 which show that BCY6136 demonstrated dose-dependent antitumor activity in the MOLP-8 xenograft multiple myeloma model and BCY6082 demonstrated significant antitumor activity (see FIGS. 32 and 33 and Tables 59 and 60).

In a further embodiment, the cancer is fibrosarcoma. Data is presented herein in Study 23 which show that BCY6173, BCY6135, BCY6174 and BCY6175 demonstrated dose dependent antitumor activity and BCY6082, BCY6031, BCY6033 and BCY6136 demonstrated potent antitumor activity in the HT-1080 xenograft fibrosarcoma model (see FIGS. 34 to 41 and Tables 61 and 62).

References herein to the term “prevention” involves administration of the protective composition prior to the induction of the disease. “Suppression” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. “Treatment” involves administration of the protective composition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available. The use of animal model systems is facilitated by the present invention, which allows the development of polypeptide ligands which can cross react with human and animal targets, to allow the use of animal models.

Furthermore, data is presented herein which demonstrates an association between copy number variation (CNV) and gene expression for EphA2 from multiple tumor types. Thus, according to a further aspect of the invention, there is provided a method of preventing, suppressing or treating cancer, which comprises administering to a patient in need thereof an effector group and drug conjugate of the peptide ligand as defined herein, wherein said patient is identified as having an increased copy number variation (CNV) of EphA2.

In one embodiment, the cancer is selected from those identified herein as having increased CNV of EphA2. In a further embodiment, the cancer is breast cancer.

The invention is further described below with reference to the following examples.

EXAMPLES

Precursor Precursor Abbreviations Name Name CAS Supplier 1Nal 1-Naphthylalanine Fmoc-3-(1-naphthyl)-L- 96402- Fluorochem alanine 49-2 2FuAla 2-Furylalanine Fmoc-L-2-furylalanine 159611- Combi 02-6 Blocks 2Nal 2-Naphthylalanine Fmoc-3-(2-naphthyl)-L- 112883- Alfa Aesar alanine 43-9 3,3-DPA 3,3-Diphenylalanine fmoc-3,3-diphenylalanine 189937- Alfa Aesar 46-0 3,4-DCPhe 3,4- Fmoc-3,4-dichloro-L- 17766- PolyPeptide Dichlorophenylalanine phenylalanine 59-5 3Pal 3-(3-Pyridyl)- N-Fmoc-3-(3-pyridyl)- 175453- Fluorochem Alanine Lβnine 07-3 4,4-BPA 4,4′-Biphenylalanine Fmoc-L-4,4′- 199110- Alfa Aesar Biphenylalanine 64-0 4BenzylPro 4-Benzyl- Fmoc-4-Benzyl- PolyPeptide pyrrolidine-2- pyrrolidine-2-carboxylic carboxylic acid acid 4BrPhe 4- Fmoc-4-Bromo-L- 198561- PolyPeptide Bromophenylalanine phenylalanine 04-5 4FlPro 4-Fluoro-pyrrolidine- Fmoc-4-fluoro-pyrrolidine- 203866- PolyPeptide 2-carboxylic acid 2-carboxylic acid 19-7 4MeoPhe 4- Fmoc-4- 77128- Iris Biotech Methoxyphenylalanine Methoxyphenylalanine 72-4 4Pal 3-(4-Pyridyl)- N-Fmoc-3-(4-pyridyl)-L- 169555- Fluorochem Alanine alanine 95-7 4PhenylPro 4-Phenyl- Fmoc-4-phenyl- 269078- Cambridge pyrrolidine-2- pyrrolidine-2-carboxylic 71-9 Bioscience carboxylic acid acid Ac Acetyl AC3C 1- 1-(Fmoc- 126705- Iris Biotech Aminocyclopropane- amino)cyclopropanecar- 22-4 1-carboxylic acid boxylic acid AC4C 1-Amino-1- 1-(Fmoc-amino)- 885951- Fluorochem cyclobutanecar- cyclobutylcarboxylic acid 77-9 boxylic acid AC5C 1-Amino-1- 1-(Fmoc- 117322- Iris Biotech cyclopentanecar- amino)cyclopentanecar- 30-2 boxylic acid boxylic acid AF488 AlexaFluor488 AlexaFluor488-NHS Ester Fisher Scientific Aib 2-Aminoisobutyric Fmoc-α-aminoisobutyric 94744- Fluorochem acid acid 50-0 Aza-Gly Azaglycine Aze Azetidine Fmoc-L-azetidine-2- 136552- Combi carboxylic acid 06-2 Blocks β-Ala β-Alanine Fmoc-β-alanine 35737- Fluorochem 10-1 β-AlaSO₃H β-Alanine(SO₃H) Fmoc-alpha-sulfo-beta- 1005412- Iris Biotech Alanine 03-2 C5g Cyclopentylglycine Fmoc-L- 220497- Fluorochem cyclopentylglycine 61-0 Cba β-Cyclobutylalanine Fmoc-β-cyclobutyl-L- 478183- IRIS alanine 62-9 Biotech GmbH Cpa β- Fmoc-β-cyclopropyl-L- 214750- Fluorochem Cyclopropylalanine alanine 76-2 Cpg Cyclopropylglycine Fmoc-L- 1212257- Apollo cycloproprylglycine 18-5 Scientific Cya Cysteic acid Fmoc-L-cysteic acid 320384- 09-6 D-3,3-DPA 3,3-diphenyl-D- Fmoc-3,3-diphenyl-D- 189937- Chem- alanine alanine 46-0 Impex international D-Arg D-Arginine Fmoc-D-Arginine(Pbf) 187618- Iris Biotech 60-6 D-Asp D-Aspartic acid Fmoc-D-aspartic acid 4- 112883- Sigma tert-butyl ester 39-3 aldrich D-Cya D-cysteic acid Fmoc-D-cysteic acid Costom synthesis D-K D-Lysine Fmoc-D-Lysine(Boc) 92122- Sigma 45-7 Aldrich DOTA 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid Fl 5(6)- Sigma carboxyfluorescein HArg HomoArginine Fmoc-L-HomoArg(Pbf)- 401915- Fluorochem OH 53-5 HPhe HomoPhenylalanine Fmoc-L- 132684- Iris Biotech Homophenylalanine 59-4 HyP Hydroxyproline Fmoc- 122996- Sigma Hydroxyproline(tBu)-OH 47-8 hSerMe HomoSerine(methyl) Fmoc-O-methyl-L- 173212- Iris Biotech homoserine 86-7 Lys(Dde) Lysine(Dde) N-α-Fmoc-N-ε-1-(4,4- 150629- Sigma dimethyl-2,6- 67-7 Aldrich dioxocyclohex-1- ylidene)ethyl-L-lysine NO2Phe 4- Fmoc-4-nitro-L- 95753- PolyPeptide Nitrophenylalanine phenylalanine 55-2 Phg Phenylglycine Fmoc-L-phenylglycine 102410- Combi 65-1 Blocks Pip Pipecolic acid Fmoc-L-Pipecolic acid 86069- Peptech 86-5 Sar Sarcosine, such Fmoc-Sarcosine-OH 77128- Sigma that Sar_(x) represents 70-2 x Sar residues tBuGly Tert-leucine Fmoc-L-tert-leucine 132684- Fluorochem 60-7 Thi 2-Thienylalanine Fmoc-2-Thienylalanine 130309- Novabiochem 35-2 ThiAz 3-(1,2,4-triazol-1- Fmoc-3-(1,2,4-triazol-1- 1217449- Sigma yl)-Alanine yl)-Ala-OH 37-0 ΨAla Reduced amide on backbone

Materials and Methods

Peptide Synthesis

Peptides were synthesized by solid phase synthesis. Rink Amide MBHA Resin was used. To a mixture containing Rink Amide MBHA (0.4-0.45 mmol/g) and Fmoc-Cys(Trt)-OH (3.0 eq) was added DMF, then DIC (3 eq) and HOAt (3 eq) were added and mixed for 1 hour. 20% piperidine in DMF was used for deblocking. Each subsequent amino acid was coupled with 3 eq using activator reagents, DIC (3.0 eq) and HOAT (3.0 eq) in DMF. The reaction was monitored by ninhydrin color reaction or tetrachlor color reaction. After synthesis completion, the peptide resin was washed with DMF×3, MeOH×3, and then dried under N₂ bubbling overnight. The peptide resin was then treated with 92.5% TFA/2.5% TIS/2.5% EDT/2.5% H₂O for 3 h. The peptide was precipitated with cold isopropyl ether and centrifuged (3 min at 3000 rpm). The pellet was washed twice with isopropyl ether and the crude peptide was dried under vacuum for 2 hours and then lyophilised. The lyophilised powder was dissolved in of ACN/H₂O (50:50), and a solution of 100 mM TATA in ACN was added, followed by ammonium bicarbonate in H₂O (1M) and the solution mixed for 1 h. Once the cyclisation was complete, the reaction was quenched with 1M aq. Cysteine hydrochloride (10 eq relative to TATA), then mixed and left to stand for an hour. The solution was lyophilised to afford crude product. The crude peptide was purified by Preparative HPLC and lyophilized to give the product

All amino acids, unless noted otherwise, were used in the L-configurations.

8.0 g of resin was used to generate 2.1 g BCY6099 (99.2% purity; 16.3% yield) as a white solid.

BCY6099 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 15-45% B over 20 minutes, then 3 min 95% B Retention Time: 11.31 min LCMS (ESI): m/z 1061.8 [M + 3H]³⁺, 796.5 [M + 4H]⁴⁺ Peptide mw 3183.68

4.79 g of resin was used to generate 1.07 g BCY6014 (Q1: 131.9 mg, 97.99% purity; Q2: 141.7 mg, 99.04% purity; Q3: 800.7 mg, 92.35% purity; 16.9% yield) as white a solid.

BCY6014 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 9.95 min LCMS (ESI): m/z 1013.8 [M + 3H]³⁺, 760.4 [M + 4H]⁴⁺ Peptide mw 3039.53

4.44 g of resin was used to generate 700 mg BCY6104 (95.87% purity, 10.5% yield) as white a solid.

BCY6104 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 7.06 min LCMS (ESI): m/z 1062.1 [M + 3H]³⁺, 796.8 [M + 4H]⁴⁺ Peptide mw 3185.65

4.44 g of resin was used to generate 700 mg BCY6103 (98.9% purity, 11.1% yield) as white a solid.

BCY6103 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1%TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150*4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 8.02 min LCMS (ESI): m/z 1039.1 [M + 3H]3+, 779.5 [M + 4H]4+ Peptide mw 3117.55

4.44 g of resin was used to generate 700 mg BCY6101 (95.9% purity, 10.9% yield) as white a solid.

BCY6101 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 9.79 min LCMS (ESI): m/z 1023.6 [M + 3H]³⁺, 768.0 [M + 4H]⁴⁺ Peptide mw 3069.55

4.44 g of resin was used to generate 900 mg BCY6102 (95.9% purity, 14.1% yield) as white a solid.

BCY6102 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 9.89 min LCMS (ESI): m/z 1018 [M + 3H]³⁺, 763.9 [M + 4H]⁴⁺ Peptide mw 3053.56

4.44 g of resin was used to generate 900 mg BCY6139 (97.4% purity, 11.2% yield) as white a solid.

BCY6139 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 8.95 min LCMS (ESI): m/z 1014.6 [M + 3H]³⁺, 761.2 [M + 4H]⁴⁺ Peptide mw 3042.51

1.11 g of resin was used to generate 200 mg BCY6138 (95.2% purity, 12.2% yield) as white a solid.

BCY6138 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 20 minutes, then 3 min 95% B Retention Time: 14.46 min LCMS (ESI): m/z 1037.6 [M + 3H]³⁺ Peptide mw 3111.63

4.44 g of resin was used to generate 600 mg BCY6137 (98.9% purity, 9.06% yield) as white a solid.

BCY6137 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 14.46 min LCMS (ESI): m/z 1092.7 [M + 3H]³⁺, 819.6 [M + 4H]⁴⁺ Peptide mw 3275.8

1.11 g of resin was used to generate 99.2 mg BCY6042 (99.2% purity, 7.0% yield) as white a solid.

BCY6042 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 9.12 min LCMS (ESI): m/z 943.5 [M + 3H]³⁺ Peptide mw 2825.31

4.79 g of resin was used to generate 732.0 mg BCY6019 (92.82% purity, 12.2% yield) as white a solid.

BCY6019 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 11.36 min LCMS (ESI): m/z 935.5 [M + 3H]³⁺ Peptide mw 2805.32

To a solution of BCY6019 (0.05 g, 17.82 μmol, 1.00 eq) in H₂O (3 mL) was adjusted PH=11 by Na₂CO₃ (aq) and added acetyl acetate (5.46 mg, 53.46 μmol, 5.01 μL, 3.00 eq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed BCY6019 was consumed completely and one main peak with desired MS was detected. The reaction was adjusted PH=7 by 1 N HCl and directly purified by prep-HPLC (TFA condition). Compound BCY6059 (18.1 mg, 6.36 μmol, 35.67% yield) was obtained as a white solid.

BCY6059 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 6.67 min LCMS (ESI): m/z 949.8 [M + 3H]³⁺ Peptide mw 2848.36

1.11 g of resin was used to generate 45.2 mg BCY6160 (95.5% purity, 2.5% yield) as white a solid.

BCY6160 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 11.38 min LCMS (ESI): m/z 1124.9 [M + 3H]³⁺ Peptide mw 3376.83

4.79 g of resin was used to generate 2.42 g BCY6009 (>88.92% purity, 36.0% yield) as white a solid.

BCY6009 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 10.16 min LCMS (ESI): m/z 1008.9 [M + 3H]³⁺, 756.9 [M + 4H]⁴⁺ Peptide mw 3025.5

1.19 g of resin was used to generate 189.9 mg BCY6017 (95.05% purity, 16.8% yield) as white a solid.

BCY6017 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 10.01 min LCMS (ESI): m/z 1129.1 [M + 2H]²⁺, 753.0 [M + 3H]³⁺ Peptide mw 2257.67

1.19 g of resin was used to generate 289.1 mg BCY6018 (97.92% purity, 21.0% yield) as white a solid.

BCY6018 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 9.77 min LCMS (ESI): m/z 1342.9 [M + 2H]²⁺, 895.3 [M + 3H]³⁺ Peptide mw 2684.14

1.11 g of resin was used to generate 150.0 mg BCY6152 (98.75% purity; 9.5% yield) as white a solid.

BCY6152 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 10.09 min LCMS (ESI): m/z 1040.3 [M + 3H]³⁺ Peptide mw 3119.59

1.11 g of resin was used to generate 120.0 mg BCY6141 (97.91% purity; 7.3% yield) as white a solid.

BCY6141 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 11.41 min LCMS (ESI): m/z 1085.5 [M + 3H]³⁺ Peptide mw 3255.78

1.11 g of resin was used to generate 285.0 mg BCY6026 (97.7% purity; 24.2% yield) as white a solid.

BCY6026 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 9.31 min LCMS (ESI): m/z 1150 [M + 2H]²⁺, 767.0 [M + 3H]³⁺ Peptide mw 2299.71

1.11 g of resin was used to generate 140.0 mg BCY6153 (98.59% purity; 9.9% yield) as white a solid.

BCY6153 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 20-50% B over 20 minutes, then 3 min 95% B Retention Time: 10.33 min LCMS (ESI): m/z 1382.6 [M + 2H]²⁺, 922.0 [M + 3H]³⁺ Peptide mw 2764.2

Preparation of Bicyclic Peptide Drug Conjugates

The general schematic for preparing Bicycle drug conjugates (BDCs) is shown in FIG. 3 and Table A describes the component targeting bicycle and linker/toxin within each BDC.

TABLE A BDC Targeting (BCY Bicycle No) (BCY No) Linker/Toxin 6136 6099 ValCit-MMAE 6033 6014 6029 6009 6122 6104 6053 6018 6049 6017 6037 6019 6030 6009 TrpCit-MMAE 6034 6014 6050 6017 6054 6018 6038 6019 6061 6014 ValLys-MMAE 6174 6099 6062 6014 D-TrpCit-MMAE 6135 6099 DM1-SS— 6031 6014 6134 6104 6027 6009 6047 6017 6035 6019 6051 6018 6154 6152 6155 6153 6173 6099 DM1-SS(SO3H)— 6082 6014 6150 6018 6151 6104 6162 6138 6161 6137 6032 6014 DM1-SS—(Me)— 6052 6018 6048 6017 6036 6019 6028 6009 6039 6014 DM1-(Me)—SS—(Me)— 6055 6014 DM1-SS—(Me2)— 6077 6014 DM1-SS—(Me)—SO3H— 6063 6014 Non-cleavable (MMAE) 6064 6014 Non-cleavable (DM1) 6105 6014 MMAE-Ala-Ala-Asn 6106 6014 MMAE-D-Ala-Phe- 6175 6099 Lys- 6107 6014 MMAE-Glu-Pro-Cit- Gly-hPhe-Tyr-Leu-

The synthesis of Bicyclic Peptide Drug Conjugates BCY6027, BCY6028, BCY6031 and BCY6032 listed in Table 6 were performed using the protocol disclosed in WO 2016/067035.

Activated Bicycle Peptides with Formula (C) and (D):

were synthesised by reacting the free amino group of the bicycle precursors with, respectively, SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate, Annova Chem) and SPDB (N-succinimidyl 3-(2-pyridyldithio)propionate, Annova Chem) in DMSO. Concentrations of bicycle precursors were 10 mM or higher, with a 1.3-fold excess of SPP or SPDB, and a 20-fold excess of diisopropylethylamine, at room temperature. The reaction was judged complete after 1 hour, as judged by LCMS. Purification was performed by reverse phase as described above. Appropriate fractions were lyophilised.

Activated bicycle peptides with formula (C) and (D) were disulphide exchanged with 1.15 equivalents of DM1 (as the free thiol), in semi aqueous conditions (50% dimethylacetamide and 50% 100 mM sodium acetate pH 5.0 supplemented with 2 mM EDTA) for 21 hours at room temperature under a nitrogen gas blanket. Concentrations of activated bicycle peptides with structure C and D in the reaction were at 10 mM or higher.

This was followed by standard reverse phase purification using a C18 column. Fractions at purity greater than 95% were isolated and lyophilised. The materials did not contain measurable quantities of free toxin.

The peptide was synthesized by solid phase synthesis. 50 g CTC Resin (sub: 1.0 mmol/g) was used. To a mixture containing CTC Resin (50 mmol, 50 g, 1.0 mmol/g) and Fmoc-Cit-OH (19.8 g, 50 mmol, 1.0 eq) was added DCM (400 mL), then DIEA (6.00 eq) was added and mixed for 3 hours. And then MeOH (50 mL) was added and mixed for 30 min for capping. 20% piperidine in DMF was used for deblocking. Boc-Val-OH (32.5 g, 150 mmol, 3 eq) was coupled with 3 eq using HBTU (2.85 eq) and DIPEA (6.0 eq) in DMF (400 mL). The reaction was monitored by ninhydrin colour reaction test. After synthesis completion, the peptide resin was washed with DMF×3, MeOH×3, and then dried under N₂ bubbling overnight. After that the peptide resin was treated with 20% HFIP/DCM for 30 min for 2 times. The solution was removed on a rotary evaporator to give the crude. The crude peptide was dissolved in ACN/H2O, then lyophilized twice to give the peptide product (17.3 g crude).

LCMS (ESI): m/z 374.9 [M + H]⁺ Molecular weight 374.44

A solution of Compound 2 (4.00 g, 10.68 mmol, 1.00 eq) in DCM (40.00 mL) and MeOH (20.00 mL) was stirred at room temperature, then (4-aminophenyl)methanol (1.58 g, 12.82 mmol, 1.20 eq) and EEDQ (5.28 g, 21.37 mmol, 2.00 eq) were added and the mixture stirred in the dark for 9 hrs. TLC (dichloromethane/methanol=5/1, Rf=0.56) indicated one new spot had formed. The reaction mixture was concentrated under reduced pressure to remove solvent. The resulting residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-20% MeOH/DCM @ 80 mL/min). Compound 3 (3.00 g, 6.26 mmol, 58.57% yield) was obtained as a white solid.

LCMS (ESI): m/z 480.1 [M + H]⁺ Molecular weight 479.58

To a solution of Compound 3 (3.00 g, 6.26 mmol, 1.00 eq) in anhydrous THF (35.00 mL) and anhydrous DCM (15.00 mL) was added (4-nitrophenyl) chloroformate (6.31 g, 31.30 mmol, 5.00 eq) and pyridine (2.48 g, 31.30 mmol, 2.53 mL, 5.00 eq), and the mixture was stirred at 25° C. for 5 hrs. TLC (dichloromethane/methanol=10/1, Rf=0.55) indicated a new spot had formed. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-10% DCM/MeOH@ 80 mL/min). Compound 4 (2.00 g, 3.10 mmol, 49.56% yield) was obtained as a white solid.

LCMS (ESI): m/z 667.3 [M + Na]⁺ Molecular weight 644.68

A mixture of Compound 4 (278.43 mg, 387.80 μmol, 1.00 eq) and DIEA (501.19 mg, 3.88 mmol, 677.29 μL, 10.00 eq) in DMF (5.00 mL) was stirred under nitrogen for 10 min. MMAE (250.00 mg, 387.80 μmol, 1.00 eq) and HOBt (52.40 mg, 387.80 μmol, 1.00 eq) were added and the mixture was stirred at 0° C. under nitrogen for 20 min and stirred at 30° C. for additional 18 hrs. LC-MS showed one main peak with desired mass was detected. The resulting mixture was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0-50% MeCN/H₂O @ 75 mL/min). Compound 5 (190.00 mg, 155.29 μmol, 40.04% yield) was obtained as a white solid.

LCMS (ESI): m/z 1223.4 [M + H]⁺ Molecular weight 1223.57

To a solution of Compound 5 (170.00 mg, 138.94 μmol, 1.00 eq) in DCM (2.70 mL) was added 2,2,2-trifluoroacetic acid (413.32 mg, 3.62 mmol, 268.39 μL, 26.09 eq), and the mixture was stirred at 25° C. for 1 hr. LC-MS showed Compound 5 was consumed completely. The mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in THF (10.00 mL) and was added K₂CO₃ (192.03 mg, 1.39 mmol, 10.00 eq), the mixture was stirred at room temperature for additional 3 hrs. LC-MS showed one main peak with desired mass was detected. The resulting reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0-50% MeCN/H₂O @ 75 mL/min). Compound 6 (110.00 mg, 97.92 μmol, 70.48% yield) was obtained as a white solid.

LCMS (ESI): m/z 1123.4 [M + H]⁺ Molecular weight 1123.45

To a solution of Compound 6 (110.00 mg, 97.92 μmol, 1.00 eq) in DMA (5 mL), DIEA (25.31 mg, 195.83 μmol, 34.20 μL, 2.00 eq) and tetrahydropyran-2,6-dione (22.34 mg, 195.83 μmol, 2.00 eq). The mixture was stirred at room temperature for 18 hrs. LC-MS showed Compound 6 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0-50% MeCN/H₂O @ 75 mL/min). Compound 7 (100.00 mg, 80.81 μmol, 82.53% yield) was obtained as a white solid.

LCMS (ESI): m/z 1237.4 [M + H]⁺ Molecular weight 1236.74

To a solution of Compound 7 (100.00 mg, 80.81 μmol, 1.00 eq) in DMA (4.5 mL) and DCM (1.5 mL) was added 1-hydroxypyrrolidine-2,5-dione (27.90 mg, 242.42 μmol, 3.00 eq) under N₂, the mixture was stirred at 0° C. for 30 min. EDCl (46.47 mg, 242.43 μmol, 3.00 eq) was added in the mixture, and the mixture was stirred at 25° C. for additional 16 hrs. LC-MS showed Compound 7 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0-50% MeCN/H₂O @ 75 mL/min). Compound 8 (90.00 mg, 60.69 μmol, 75.11% yield) was obtained as a white solid.

LCMS (ESI): m/z 1334.5 [M + H]⁺ Molecular weight 1334.62

General Procedure for Coupling MMAE-PABC-Cit-Val-Glutarate-NHS with Targeting Bicycles

To a solution of Bicycle (1.0-1.3 eq) in DMA was added DIEA (3 eq) and MMAE-PABC-Cit-Val-Glutarate-NHS (1 eq). The mixture was stirred at 25° C. for 18 hr. The reaction was monitored by LC-MS and once complete, was directly purified by preparative HPLC.

BCY6099 (71.5 mg, 22.48 μmol) was used as the bicycle reagent. Compound BCY6136 (40.9 mg, 9.05 μmol, 40.27% yield, 97.42% purity) was obtained as a white solid.

BCY6136 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 11.35 min LCMS (ESI): m/z 1468.1 [M + 3H]³⁺, 1101.2 [M + 4H]⁴⁺, 881.3 [M + 5H]⁵⁺ Peptide mw 4404.2

BCY6014 (70.00 mg, 22.47 μmol, 1.00 eq) was used as the bicycle reagent. Compound BCY6033 (33.90 mg, 7.96 μmol, 34.57% yield) was obtained as a white solid.

BCY6033 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 7.47 min LCMS (ESI): m/z 1065.2 [M + 4H]⁴⁺, 852.2 [M + 5H]⁵⁺ Peptide mw 4259.04

BCY6009 (70.0 mg, 22.47 μmol, 1 eq) was used as the bicycle reagent. Compound BCY6029 (32.9 mg, 7.75 μmol, 33.49% yield) was obtained as a white solid.

BCY6029 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 7.46 min LCMS (ESI): m/z 1061.7 [M + 4H]⁴⁺ Peptide mw 4245.02

BCY6104 (71.59 mg, 22.48 μmol, 1.00 eq) was used as the bicycle reagent. Compound BCY6122 (38.30 mg, 8.57 μmol, 38.14% yield, 98.58% purity) was obtained as a white solid.

BCY6122 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 10.72 min LCMS (ESI): m/z 1101.8 [M + 4H]⁴⁺, 881.5 [M + 5H]⁵⁺ Peptide mw 4406.18

BCY6018 (72.40 mg, 26.97 μmol, 1.2 eq) was used as the bicycle reagent. Compound BCY6053 (38.3 mg, 9.81 μmol, 43.65% yield) was obtained as a white solid.

BCY6053 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 12.95 min LCMS (ESI): m/z 1301.7 [M + 3H]³⁺, 976.5 [M + 4H]⁴⁺ Peptide mw 3905.67

BCY6017 (50.75 mg, 22.48 μmol, 1.2 eq) was used as the bicycle reagent. Compound BCY6049 (22.5 mg, 6.47 μmol, 34.54% yield) was obtained as a white solid.

BCY6049 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 14.28 min LCMS (ESI): m/z 1159.6 [M + 3H]³⁺, 869.8 [M + 4H]⁴⁺ Peptide mw 3479.2

BCY6019 (65.00 mg, 22.47 μmol, 1.00 eq) was used as the bicycle reagent. Compound BCY6037 (26.80 mg, 6.66 μmol, 28.74% yield) was obtained as a white solid.

BCY6037 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 8.79 min LCMS (ESI): m/z 1342.1 [M + 3H]³⁺, 1006.6 [M + 4H]⁴⁺ Peptide mw 4025.84

General Procedure for Preparation of 3

To a solution of compound 2 (4.00 g, 8.67 mmol, 1.00 eq), DIC (1.61 g, 12.78 mmol, 1.97 mL, 9.00 eq) and HOBt (10.54 g, 78.00 mmol, 9.00 eq) in DMF (30.00 mL) was added (4-aminophenyl)methanol (9.61 g, 78.00 mmol, 9.00 eq). The mixture was stirred at 15° C. for 1 hour. LC-MS showed compound 2 was consumed completely and one main peak with desired MS was detected. The mixture was purified by prep-HPLC. Compound 3 (4.20 g, 7.41 mmol, 85.49% yield) was obtained as a white solid.

LCMS (ESI): m/z 566.9 [M + H]⁺ Molecular weight 566.66

General Procedure for Preparation of 4

To a solution of compound 3 (4.20 g, 6.30 mmol, 1.00 eq), DIPEA (1.09 g, 8.40 mmol, 1.47 mL, 7.00 eq) in DMF (30.00 mL) was added bis(4-nitrophenyl) carbonate (11.50 g, 37.79 mmol, 6.00 eq) in one part. The mixture was stirred at 0-15° C. for 1.5 hour. LC-MS showed compound 3 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (TFA condition). Compound 4 (2.00 g, 2.40 mmol, 38.16% yield) was obtained as a white solid.

LCMS (ESI): m/z 732.0 [M + H]⁺ Molecular weight 731.76

General Procedure for Preparation of 5

To a solution of compound 4 (300.00 mg, 360.63 μmol, 1.00 eq), DIEA (93.22 mg, 721.27 μmol, 125.97 μL, 3.00 eq) in DMF (10.00 mL) was added MMAE (233.03 mg, 324.57 μmol, 0.90 eq) and HOBt (48.73 mg, 360.63 μmol, 1.00 eq) at 0° C. The mixture was stirred at 30° C. for 18 hour. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 5 (250.00 mg, 190.75 μmol, 52.89% yield) was obtained as a yellow solid.

LCMS (ESI): m/z 1310.5 [M + H]⁺ Molecular weight 1310.65

General Procedure for Preparation of 6

To a solution of compound 5 (240.00 mg, 183.12 μmol, 1.00 eq) in DCM (10.00 mL) was added TFA (1.54 g, 13.51 mmol, 1.00 mL, 73.76 eq). The mixture was stirred at 15° C. for 2 hour. And the mixture was concentrated under reduced pressure to remove solvent to give a residue, the residue was dissolved in THF and added K₂CO₃ and stirred at 15° C. for 2 h. LC-MS showed compound 5 was consumed completely and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). The crude product 6 (125.00 mg, 94.37 μmol, 51.53% yield, TFA) was used into the next step without further purification.

LCMS (ESI): m/z 1210.4 [M + H]⁺ Molecular weight 1209.53

General Procedure for Preparation of 7

To a solution of compound 6 (125.00 mg, 94.37 μmol, 1.00 eq, TFA) in DMA (5.00 mL) was added DIEA (24.39 mg, 188.75 μmol, 32.96 μL, 2.00 eq), tetrahydropyran-2,6-dione (21.54 mg, 188.75 μmol, 2.00 eq). The mixture was stirred at 15° C. for 2 hour. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 7 (100.00 mg, 75.49 μmol, 80.00% yield) was obtained as a white solid.

LCMS (ESI): m/z 1324.5 [M + H]⁺ Molecular weight 1324.63

General Procedure for Preparation of 8 (MMAE-PABC-Cit-Trp-Glutarate-NHS)

To a solution of compound 7 (100.00 mg, 75.49 μmol, 1.00 eq), 1-hydroxypyrrolidine-2, 5-dione (26.07 mg, 226.48 μmol, 3.00 eq) in DMA (3.00 mL) and DCM (1.00 mL) was added EDCl (43.42 mg, 226.48 μmol, 3.00 eq). The mixture was stirred at 15° C. for 4 hour. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected. The DCM was removed. Directly was purified by prep-HPLC (neutral condition). Compound 8 (60.00 mg, 42.20 μmol, 55.91% yield) was obtained as a white solid.

LCMS (ESI): m/z 711.2 [M + 2H]²⁺ Molecular weight 1421.7

General Procedure for Coupling MMAE-PABC-Cit-Trp-Glutarate-NHS with Targeting Bicycles

To a solution of Bicycle (1.0-1.3 eq) in DMA was added DIEA (3 eq) and MMAE-PABC-Cit-Trp-Glutarate-NHS (1 eq). The mixture was stirred at 25° C. for 18 hr. The reaction was monitored by LC-MS and once complete, was directly purified by preparative HPLC.

BCY6009 (47.29 mg, 14.07 μmol, 1.00 eq) was used as the bicycle reagent. Compound BCY6030 (0.0156 g, 3.51 μmol, 24.93% yield, 97.41% purity) was obtained as a, white solid.

BCY6030 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 7.90 min LCMS (ESI): m/z 1083.7 [M + 4H]⁴⁺ Peptide mw 4332.17

BCY6014 (88.21 mg, 23.21 μmol, 1.10 eq) was used as the bicycle reagent. Compound BCY6034 (27.70 mg, 6.05 μmol, 28.70% yield, 95.02% purity) was obtained as a white solid.

BCY6034 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 30-60% B over 20 minutes, then 3 min 95% B Retention Time: 11.49 min LCMS (ESI): m/z 1449.3 [M + 3H]³⁺, 1087.4 [M + 4H]⁴⁺ Peptide mw 4346.13

BCY6017 (57.17 mg, 25.32 μmol, 1.2 eq) was used as the bicycle reagent. Compound BCY6050 (0.0519 g, 14.56 μmol, 69.01% yield) was obtained as a white solid.

BCY6050 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 13.55 min LCMS (ESI): m/z 1188.5 [M + 3H]³⁺, 891.7 [M + 4H]⁴⁺ Peptide mw 3564.25

BCY6018 (67.97 mg, 25.32 μmol, 1.2 eq) was used as the bicycle reagent. Compound BCY6054 (40.10 mg, 10.05 μmol, 47.62% yield) was obtained as a white solid.

BCY6054 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 13.73 min LCMS (ESI): m/z 1330.4 [M + 3H]³⁺, 998.1 [M + 4H]⁴⁺ Peptide mw 3990.72

BCY6019 (81.39 mg, 23.21 μmol, 1.10 eq) was used as the bicycle reagent. Compound BCY6038 (34.10 mg, 8.02 μmol, 38.00% yield, 96.68% purity) was obtained as a white solid.

BCY6038 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 9.56 min LCMS (ESI): m/z 1371.0 [M + 3H]³⁺, 1028.3 [M + 4H]⁴⁺ Peptide mw 4111.9

General Procedure for Preparation of Compound 2

To a mixture of Compound 1 (3.00 g, 5.89 mmol, 1 eq) and (4-aminophenyl)methanol (869.93 mg, 7.06 mmol, 1.2 eq) in DCM (35 mL) and MeOH (18 mL) was added EEDQ (2.91 g, 11.77 mmol, 2 eq) in the dark under nitrogen, the mixture was stirred at 25° C. for 5 hr. LC-MS showed Compound 1 was consumed completely and one main peak with desired MS was detected. The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-20% MeOH/DCM @ 80 mL/min). Compound 2 (2.2 g, 3.58 mmol, 60.79% yield) was obtained as a white solid.

LCMS (ESI): m/z 615.0 [M + H]⁺ Molecular weight 614.78

General Procedure for Preparation of Compound 3

To a solution Compound 2 (500 mg, 813.31 μmol, 1 eq) in THF (10 mL) was added DIEA (630.69 mg, 4.88 mmol, 849.98 μL, 6 eq) at 0° C. under nitrogen with stirring for 30 mins. Then bis(4-nitrophenyl) carbonate (1.48 g, 4.88 mmol, 6 eq) was added thereto, the mixture was stirred at 25° C. under nitrogen for additional 21 hr. LC-MS showed one main peak with desired MS was detected. The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-20% MeOH/DCM @ 40 mL/min). Compound 3 (500 mg, 641.13 μmol, 78.83% yield) was obtained as a yellow solid.

LCMS (ESI): m/z 780.0 [M + H]⁺ Molecular weight 779.89

General Procedure for Preparation of Compound 4

To a mixture of Compound 3 (500 mg, 512.90 μmol, 1.23 eq) in DMF (8 mL) was added DIEA (135.01 mg, 1.04 mmol, 181.95 μL, 2.5 eq) with stirring at 0° C. for 30 mins. Then MMAE (300 mg, 417.84 μmol, 1 eq) and HOBt (84.69 mg, 626.76 μmol, 1.5 eq) was added thereto, and the mixture was stirred at 40° C. for 15 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired MS was detected. The residue was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0-60% MeCN/H2O @ 75 mL/min). Compound 4 (330 mg, 242.87 μmol, 58.13% yield) was obtained as a white solid.

LCMS (ESI): m/z 679.7 [M + 2H]²⁺ Molecular weight 1358.77

General Procedure for Preparation of Compound 5

To a solution of Compound 4 (325 mg, 239.19 μmol, 1 eq) in DCM (18 mL) was added TFA (3.03 g, 26.60 mmol, 1.97 mL, 111.22 eq) at 0° C., the mixture was stirred at 25° C. for 2 hr. LC-MS showed compound 4 was consumed completely. Then the reaction mixture was concentrated under reduced pressure to give a residue, the residue was dissolved in THF (10 mL) and K₂O₃ (661.16 mg, 4.78 mmol, 20 eq) was added thereto. The mixture was stirred at 25° C. for 15 hrs. LC-MS showed one main peak with desired MS was detected. The resulting reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0-60% MeCN/H2O @ 75 mL/min). Compound 5 (170 mg, 135.07 μmol, 56.47% yield) was obtained as a white solid.

LCMS (ESI): m/z 629.7 [M + 2H]²⁺ Molecular weight 1258.65

General Procedure for Preparation of Compound 6

A round bottle containing a solution of compound 5 (140 mg, 111.23 μmol, 1 eq) in DMA (5 mL) was purged using a nitrogen balloon and added DIEA (28.75 mg, 222.46 μmol, 38.75 μL, 2 eq) at 0° C. with stirring for 10 mins, tetrahydropyran-2,6-dione (25.38 mg, 222.46 μmol, 2 eq) was added as a solution in DMA. The mixture was stirred at 25° C. for 12 hr. LC-MS showed compound 5 was consumed completely and one main peak with desired MS was detected. The resulting reaction mixture was purified by flash C18 gel chromatography (ISCO®; 43 g SepaFlash® C18 Flash Column, Eluent of 0-60% MeCN/H2O @40 mL/min). Compound 6 (120 mg, 87.42 μmol, 78.59% yield) was obtained as a white solid.

LCMS (ESI): m/z 686.7 [M + 2H]²⁺ Molecular weight 1372.75

General Procedure for Preparation of Compound 7 (MMAE-PABC-Lys(Dde)-Val-Glutarate-NHS)

To a solution of compound 6 (120 mg, 87.42 μmol, 1 eq) in DMA (9 mL) and DCM (3 mL) was added 1-hydroxypyrrolidine-2,5-dione (30.18 mg, 262.25 μmol, 3 eq) with stirring, and EDCl (50.27 mg, 262.25 μmol, 3 eq) was added thereto, the mixture was stirred at 0° C. for 30 mins and at 25° C. for additional 19 hr. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected. The resulting reaction mixture was concentrated under reduced pressure to remove DCM. The mixture was purified by flash C18 gel chromatography (ISCO®; 43 g SepaFlash® C18 Flash Column, Eluent of 0-60% MeCN/H2O @ 40 mL/min). Compound 7 (60 mg, 40.82 μmol, 46.70% yield) was obtained as a white solid.

LCMS (ESI): m/z 735.3 [M + 2H]²⁺ Molecular weight 1469.83

General Procedure for Coupling MMAE-PABC-Lys(Dde)-Val-Glutarate-NHS with Targeting Bicycles

To a solution of Bicycle (1.0-1.3 eq) in DMA was added DIEA (3 eq) and MMAE-PABC-Lys(Dde)-Val-Glutarate-NHS (1 eq). The mixture was stirred at 25° C. for 18 hr. The reaction was monitored by LC-MS and once complete, was directly purified by preparative HPLC.

General Procedure for Dde Deprotection

To a solution of Ode protected peptide (1 eq) in DMF was added hydrazine hydrate (6500 eq), and the mixture was stirred at 25° C. for 1 hr. LC-MS was used to monitor the reaction, and once complete, the mixture was purified by preparative HPLC and the clean fractions lyophilised.

BCY6014 (124.12 mg, 40.82 μmol, 1.2 eq) was used as the bicycle reagent. Dde-BCY6038 (80 mg, 18.20 μmol, 53.51% yield) was obtained as a white solid.

LCMS (ESI): m/z 1099.0 [M + 4H]⁴⁺, 879.4 [M + 5H]⁵⁺ Molecular weight 4395.24

Dde-BCY6061 (78 mg, 17.75 μmol) was deprotected using hydrazine according to the general procedure to give BCY6061 (47.1 mg, 11.13 μmol, 62.73% yield) as a white solid.

BCY6061 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 12.01 min LCMS (ESI): m/z 1058.1 [M + 4H]⁴⁺, 846.5 [M + 5H]⁵⁺ Peptide mw 4230.03

BCY6099 (389.77 mg, 122.47 μmol, 1.2 eq) was used as the bicycle reagent. Dde-BCY6174 (0.250 g, 55.10 μmol, 53.99% yield) was obtained as a white solid.

LCMS (ESI): m/z 1513.0 [M + 3H]³⁺, 1135.0 [M + 4H]⁴⁺, 908.2 [M + 5H]⁵⁺ Molecular weight 4538.38

Dde-BCY6174 (0.250 g, 55.10 μmol, 1.0 eq) was deprotected using hydrazine according to the general procedure to give BCY6174 (0.1206 g, 27.45 μmol, 49.82% yield) as a white solid.

BCY6174 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 9.85 min LCMS (ESI): m/z 1458.5 [M + 3H]³⁺, 1094.1 [M + 4H]⁴⁺, 875.4 [M + 5H]⁵⁺ Peptide mw 4373.17

General Procedure for Preparation of Compound 3

To a solution of compound 1 (2 g, 4.33 mmol, 1.00 eq), DIC (4.92 g, 39.00 mmol, 6.00 mL, 9.00 eq), HOBt (5.27 g, 39.00 mmol, 9.00 eq) in DMF (30.00 mL) was added (4-aminophenyl)methanol (4.80 g, 39.00 mmol, 9.00 eq). The mixture was stirred at 15° C. for 1 hour. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 2 (2 g, 3.53 mmol, 81.45% yield) was obtained as a white solid.

LCMS (ESI): m/z 666.9 [M + H]⁺ Molecular weight 666.78

General Procedure for Preparation of Compound 4

To a solution of compound 2 (2 g, 3.00 mmol, 1 eq), DIEA (2.71 g, 21.00 mmol, 3.66 mL, 7 eq) in DMF (20 mL) was added bis(4-nitrophenyl) carbonate (5.48 g, 18.00 mmol, 6 eq) in one part at 0° C. The mixture was stirred at 0-15° C. for 2 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 3 (0.9 g, 1.08 mmol, 36.07% yield) was obtained as a yellow solid.

LCMS (ESI): m/z 832.0 [M + H]⁺ Molecular weight 831.88

General Procedure for Preparation of Compound 5

To a solution of compound 3 (350 mg, 420.74 μmol, 1.00 eq), HOBt (56.85 mg, 420.74 μmol, 1 eq) and DIEA (163.13 mg, 1.26 mmol, 219.86 μL, 3 eq) in DMF (10 mL) was added MMAE (302.08 mg, 420.74 μmol, 1 eq) at 0° C. The mixture was stirred at 40° C. for 18 hour. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 4 (0.22 g, 155.95 μmol, 37.06% yield) was obtained as a yellow solid.

LCMS (ESI): m/z 1410.5 [M + H]⁺ 705.7 [M + 2H]²⁺ Molecular weight 1410.76

General Procedure for Preparation of Compound 6

To a solution of compound 4 (0.21 g, 148.86 μmol, 1 eq) in DCM (9 mL) was added TFA (1.54 g, 13.51 mmol, 1 mL, 90.73 eq). The mixture was stirred at 15° C. for 4 h, and concentrated under reduced pressure to give a residue, dissolved in THF, then added K₂CO₃(s) and stirred at 15° C. for 16 h. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 5 (0.13 g, 102.02 μmol, 68.54% yield, 95% purity) was obtained as a white solid.

LCMS (ESI): m/z 1210.4 [M + H]⁺, 605.8 [M + 2H]²⁺ Molecular weight 1210.53

General Procedure for Preparation of Compound 7

To a solution of compound 5 (0.12 g, 99.13 μmol, 1 eq) in DMA (5 mL) was added DIEA (38.44 mg, 297.40 μmol, 51.80 μL, 3 eq) and tetrahydropyran-2,6-dione (22.62 mg, 198.26 μmol, 2 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 5 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 6 (0.09 g, 67.94 μmol, 68.54% yield) was obtained as a white solid.

LCMS (ESI): m/z 662.7 [M + 2H]²⁺ Molecular weight 1324.63

General Procedure for Preparation of Compound 8

To a solution of compound 6 (0.09 g, 67.95 μmol, 1 eq), HOSu (23.46 mg, 203.84 μmol, 3 eq) in DMA (6 mL) and DCM (2 mL) was added EDCl (39.08 mg, 203.84 μmol, 3 eq). The mixture was stirred at 15° C. for 16 h. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected. DCM was removed and directly purified by prep-HPLC (neutral condition). Compound 7 (0.06 g, 40.09 μmol, 59.01% yield, 95% purity) was obtained as a white solid.

LCMS (ESI): m/z 711.2 [M + 2H]²⁺ Molecular weight 1421.7

BCY6062

To a solution of BCY6014 (76.99 mg, 25.32 μmol, 1.2 eq) in DMA (5 mL) was added DIEA (8.18 mg, 63.31 μmol, 11.03 μL, 3 eq), compound 7 (0.03 g, 21.10 μmol, 1 eq). The mixture was stirred at 15° C. for 16 hr. The reaction was monitored by LC-MS and once complete, the mixture was purified by preparative HPLC. BCY6062 (0.0255 g, 5.70 μmol, 27.01% yield, 97.15% purity) was obtained as a white solid.

BCY6062 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 13.15 min LCMS (ESI): m/z 1449.2 [M + 3H]³⁺, 1087.0 [M + 4H]⁴⁺ Peptide mw 4346.13

To a solution of 2-(2-pyridyldisulfanyl)pyridine (12.37 g, 56.18 mmol, 1.50 eq in EtOH (100.00 m was added 4-sulfanylbutanoic acid (4.50 g, 37.45 mmol, 1.00 eq). The mixture was stirred at 15° C. for 18 hours under N₂. LC-MS showed compound 1 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by preparative HPLC (C18 360 g, neutral condition). Compound SPDB (1.9 g, 8.29 mmol, 22.12% yield) was obtained as a yellow solid.

¹H NMR: ES6446-8-P1A 400 MHz CDCl₃

δ ppm 1.98 (q, J=7.09 Hz, 2H), 2.45 (t, J=7.15 Hz, 2H), 2.79 (t, J=7.03 Hz, 2H), 7.03 (dd, J=7.15, 4.89 Hz, 1H), 7.19 (s, 1H), 7.56-7.65 (m, 2H), 8.41 (d, J=4.52 Hz, 1H).

LCMS (ESI): m/z 230.0 [M + H]⁺ Molecular weight 229.31

A mixture of DM1 (250.00 mg, 338.62 μmol, 1.00 eq) and 4-(2-pyridyldisulfanyl)butanoic acid (100.95 mg, 440.21 μmol, 1.30 eq) was added under nitrogen in a 50 mL of flask with DMF (10.00 mL) purged with N₂ for 30 mins. The mixture was stirred at room temperature for 1 hr. LC-MS showed that the DM1 was consumed completely and one main peak with desired mass was detected. The residue was purified by flash C18 gel chromatography (ISCO®; 120 g SepaFlash® C18 Flash Column, Eluent of 0-60% MeCN/H₂O @ 85 mL/min). DM1-SPDB (120.00 mg, 140.11 μmol, 41.38% yield) was obtained as a white solid.

LCMS (ESI): m/z 838.0 [M + H—H₂O]⁺ Molecular weight 856.44

To a solution of DM1-SPDB (120.00 mg, 140.11 μmol, 1.00 eq) and 2,3,5,6-tetrafluorophenol (69.81 mg, 420.34 μmol, 3.00 eq) in DCM (1.00 mL) and DMA (3.00 mL) was added EDCl (80.58 mg, 420.34 μmol, 3.00 eq). The mixture was stirred at 15° C. for 4 hours. LC-MS showed DM1-SPDB was consumed completely and one main peak with desired mass was detected. The DCM was removed and the residue The mixture was directly purified by preparative HPLC (neutral condition). Compound DM1-SPDB-TFP (60.00 mg, 59.73 μmol, 42.63% yield) was obtained as a white solid.

LCMS (ESI): m/z 985.9 [M + H—H₂O]⁺ Molecular weight 1004.5

General Procedure for Coupling DM1-SPDB-TFP with Targeting Bicycles

To a solution of targeting Bicycle (1.1-1.3 eq) in DMA was added DIEA (3 eq) and DM1-SPDB-TFP (1 eq). The mixture was stirred at 25° C. for 18 hr. The reaction was monitored by LC-MS and once complete, the mixture was directly purified by preparative HPLC.

BCY6099 (114.1 mg, 35.84 μmol) was used as the bicycle reagent. 22.4 mg Compound BCY6135 (5.30 μmol, 17.74% yield, 95.14% purity) was obtained as a white solid.

BCY6135 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 9.81 LCMS (ESI): m/z 1341.5 [M + 3H]³⁺, 805.0 [M + 5H]⁵⁺ Peptide mw 4021.08

BCY6014 (121.07 mg, 39.82 μmol) was used as the bicycle reagent. 59.90 mg compound BCY6031 (14.67 μmol, 36.85% yield, 95.02% purity) was obtained as a white solid.

BCY6031 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 6.284 min LCMS (ESI): m/z 1286.4 [M + 3H—H2O]³⁺, 965.6 [M + 4H—H2O]⁴⁺ Peptide mw 3877.96

BCY6104 (95.11 mg, 29.87 μmol, 1 eq) was used as the bicycle reagent. BCY6134 (0.0232 g, 5.64 μmol, 18.89% yield, 97.82% purity) was obtained as a white solid.

BCY6134 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 9.10 min LCMS (ESI): m/z 1001.8 [M + 4H—H2O]⁴⁺ Peptide mw 4026.1

BCY6009 (60.24 mg, 19.91 μmol, 1.00 eq) was used as the bicycle reagent. BCY6027 (20.40 mg, 5.11 μmol, 25.69% yield, 96.88% purity) was obtained as a white solid.

BCY6027 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 5.97 min LCMS (ESI): m/z 1932.1 [M + 2H]²⁺, 1282.5 [M + 3H—H2O]³⁺ Peptide mw 3863.99

BCY6017 (61.81 mg, 27.38 μmol, 1.1 eq) was used as the bicycle reagent. BCY6047 (0.032 g, 10.34 μmol, 41.53% yield) was obtained as a white solid.

BCY6047 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 38-68% B over 30 minutes, then 3 min 95% B Retention Time: 12.28 min LCMS (ESI): m/z 1026.3 [M + 3H—H2O]³⁺ Peptide mw 3096.1

BCY6019 (115.22 mg, 32.86 μmol, 1.10 eq) was used as the bicycle reagent. BCY6035 (37.80 mg, 10.37 μmol, 34.73% yield) was obtained as a white solid.

BCY6035 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 12.28 min LCMS (ESI): m/z 1208.8 [M + 3H—H2O]³⁺, 911.5 [M + 4H]⁴⁺ Peptide mw 3643.73

BCY6018 (73.48 mg, 27.38 μmol, 1.1 eq) was used as the bicycle reagent. BCY6051 (0.0582 g, 16.52 μmol, 66.39% yield) was obtained as a white solid.

BCY6051 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 11.37 min LCMS (ESI): m/z 880.5 [M + 4H]⁴⁺ Peptide mw 3522.57

BCY6152 (93.17 mg, 29.87 μmol, 1 eq) was used as the bicycle reagent. BCY6154 (40.10 mg, 9.93 μmol, 33.27% yield, 98.06% purity) was obtained as a white solid.

BCY6154 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 11.94 min LCMS (ESI): m/z 1313.8 [M + 3H—H2O]³⁺, 985.8 [M + 4H—H2O]⁴⁺ Peptide mw 3958.02

BCY6153 (82.55 mg, 29.87 μmol, 1 eq) was used as the bicycle reagent. BCY6155 (0.0312 g, 8.55 μmol, 28.62% yield, 98.69% purity) was obtained as a white solid.

BCY6155 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 12.93 min LCMS (ESI): m/z 897.1 [M + 4H—H2O]⁴⁺ Peptide mw 3602.63

To a solution of 4-sulfanylbutanoic acid (2.0 g, 16.64 mmol, 1 eq) and 2-(2-pyridyldisulfanyl) pyridine (11.0 g, 49.93 mmol, 3 eq) in EtOH (50 mL) was added AcOH (1.05 g, 17.48 mmol, 1 mL, 1.05 eq). The mixture was stirred at 40° C. for 16 hr under N₂. LC-MS showed one main peak with desired mass was detected and TLC indicated 4-sulfanylbutanoic acid was consumed completely. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by preparative HPLC (neutral condition). Compound 2 (2.0 g, 8.72 mmol, 52.4% yield) was obtained as yellow solid.

¹H NMR: 400 MHz CDCl₃

δ ppm 2.03-2.11 (m, 2H), 2.54 (t, J=7.20 Hz, 2H), 2.88 (t, J=7.20 Hz, 2H), 7.11-7.14 (m, 1H), 7.67-7.74 (m, 2H), 8.50 (d, J=4.80 Hz, 1H).

LCMS (ESI): 230 [M + H]⁺ Molecular weight 229.31

To a solution of compound 2 (0.5 g, 2.18 mmol, 1 eq) in DCE (5 mL) was added chlorosulfonic acid (1.5 g, 13.08 mmol, 0.89 mL, 6 eq) in three portions and DIEA (1.13 g, 8.72 mmol, 1.52 mL, 4 eq) in two portions. The mixture was stirred at 75° C. for 2 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired mass was detected. The reaction mixture was quenched by addition 3 mL of H₂O and the DCE was removed. The residue was The mixture was directly purified by preparative HPLC (neutral conditions). Compound 3 (0.68 g, 1.76 mmol, 80.6% yield, 80% purity) was obtained as light yellow oil.

¹H NMR: 400 MHz CDCl₃

δ ppm 2.49-2.54 (m, 2H), 3.63-3.67 (m, 2H), 3.90 (t, J=6.60 Hz, 2H), 7.09-7.12 (m, 1H), 7.66-7.76 (m, 2H), 8.47 (dd, J=4.80 Hz, 0.80 Hz, 1H), 8.56 (s, 1H).

LCMS (ESI): 310.0 [M + H]⁺ Molecular weight 309.37

To a solution of DM1 (1.0 g, 1.35 mmol, 1 eq) and compound 3 (502.9 mg, 1.63 mmol, 1.2 eq) in DMF (10 mL) was added NaHCO₃ (aq) until the pH reached 8. The mixture was stirred at 25° C. for 1 hr. LC-MS showed DM1 was consumed completely and one main peak with desired mass was detected. The residue was The mixture was directly purified by preparative HPLC (neutral condition). Compound DM1-SO₃H-SPDB (0.28 g, 299.0 μmol, 22.1% yield) was obtained as a white solid.

LCMS (ESI): 918.2 [M + H—H₂O]⁺ Molecular weight 936.50

To a solution of DM1-SO₃H-SPDB (103.2 mg, 896.95 μmol, 3 eq), 1-Hydroxypyrrolidine-2,5-dione (103.2 mg, 896.95 μmol, 3 eq) in DMA (6 mL) and DCM (2 mL) was added EDCl (171.9 mg, 896.95 μmol, 3 eq). The mixture was stirred at 25° C. for 16 hr. LC-MS showed DM1-SO₃H-SPDB was consumed completely and one main peak with desired mass was detected. DCM was removed. The residue was The mixture was directly purified by preparative HPLC (neutral condition). Compound DM1-SO₃H-SPDB-NHS (0.22 g, 212.85 μmol, 71.2% yield) was obtained as a white solid.

LCMS (ESI): 1015.2 [M + H—H₂O]⁺ Molecular weight 1033.57

General Procedure for Coupling DM1-SO3H-SPDB-NHS with Targeting Bicycles

To a solution of targeting Bicycle (1.1-1.3 eq) in DMA was added DIEA (3 eq) and DM1-SO3H-SPDB-NHS (1 eq). The mixture was stirred at 25° C. for 16 hr. The reaction was monitored by LC-MS and once complete, the mixture was directly purified by preparative HPLC.

BCY6099 (200.15 mg, 62.89 μmol) was used as the bicycle reagent. 57.1 mg compound BCY6173 (3.40 μmol, 22.79% yield, 95.80% purity) was obtained as a white solid.

BCY6173 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 10.30 min LCMS (ESI): m/z 1361.9 [M + 3H—H2O]³⁺, 1021.8 [M + 4H—H2O]⁴⁺ Peptide mw 4101.15

BCY6014 (711.9 mg, 234.14 μmol) was used as the bicycle reagent. 308 mg compound BCY6082 (74.97 μmol, 35.2% yield, 96.36% purity) was obtained as a white solid.

BCY6082 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 11.95 min LCMS (ESI): m/z 1299.3 [M + 3H—H2O]³⁺, 975.0 [M + 4H—H2O]⁴⁺ Peptide mw 3911.04

BCY6018 (77.91 mg, 29.03 μmol, 1 eq) was used as the bicycle reagent. BCY6150 (0.0249 g, 6.61 μmol, 22.78% yield) was obtained as a white solid.

BCY6150 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 12.31 min LCMS (ESI): m/z 1195.4 [M + 3H—H2O]³⁺ Peptide mw 3602.63

BCY6104 (120.17 mg, 37.73 μmol, 1.3 eq) was used as the bicycle reagent. BCY6151 (0.0256 g, 6.16 μmol, 21.22% yield) was obtained as a white solid.

BCY6151 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 8.68 min LCMS (ESI): m/z 1362.3 [M + 3H—H2O]³⁺ Peptide mw 4105.16

BCY6138 (82.80 mg, 26.61 μmol, 1.1 eq) was used as the bicycle reagent. BCY6162 (0.0362 g, 8.98 μmol, 37.13% yield) was obtained as a white solid.

BCY6162 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 21.09 min LCMS (ESI): m/z 1323.5 [M + 3H—H2O − 44]³⁺ Peptide mw 4026.74

BCY6137 (79.67 mg, 24.48 μmol, 1.1 eq) was used as the bicycle reagent. BCY6161 (0.0232 g, 5.26 μmol, 21.76% yield) was obtained as a white solid.

BCY6161 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 10.22 min LCMS (ESI): m/z 1392 [M + 3H—H2O]³⁺ Peptide mw 4192.33

To a solution of 2-(2-pyridyldisulfanyl)pyridine (2.46 g, 11.18 mmol, 1.50 eq) and AcOH (1.05 g, 17.49 mmol, 1.00 mL, 2.35 eq) in EtOH (50.00 mL) was added 4-sulfanylpentanoic acid (1.00 g, 7.45 mmol, 1.00 eq). The mixture was stirred at 40° C. for 18 hours under N₂. LC-MS showed compound 1 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by preparative HPLC (neutral condition). Compound SPP (1.61 g, 6.62 mmol, 88.81% yield) was obtained as a yellow solid.

¹H NMR: 400 MHz DMSO-de

δ ppm 1.36 (d, J=6.78 Hz, 3H), 1.88-2.07 (m, 2H), 2.56 (td, J=7.53, 1.76 Hz, 2H), 3.00-3.09 (m, 1H), 7.11 (ddd, J=7.34, 4.96, 1.00 Hz, 1H), 7.66 (td, J=7.78, 1.76 Hz, 1H), 7.73-7.77 (m, 1H), 8.48 (dt, J=4.02, 0.88 Hz, 1H).

LCMS (ESI): 243.8 [M + H]⁺ Molecular weight 243.34

A solution of DM1 (200 mg, 270.90 μmol, 1.00 eq), 4-(2-pyridyldisulfanyl)pentanoic acid (98.89 mg, 406.35 μmol, 1.50 eq) in H₂O (5.00 mL) was adjusted PH=8 using NaHCO₃ (aq). The mixture was stirred at 15° C. for 1 hour. LC-MS showed DM1 was consumed completely and one main peak with desired mass was detected (main MS was M+1-18). The mixture was directly purified by preparative HPLC (neutral condition). Compound DM1-SPP (120 mg, 137.86 μmol, 50.89% yield) was obtained as a white solid.

LCMS (ESI): 852.0 [M + H—H₂O]⁺ Molecular weight 870.47

To a solution of DM1-SPP (0.175 g, 201.04 μmol, 1.0 eq), 2,3,5,6-tetrafluorophenol (100.16 mg, 603.13 μmol, 3.0 eq) in DCM (1.0 mL) and DMA (3.0 mL) was added EDCl (115.62 mg, 603.13 μmol, 3.0 eq). The mixture was stirred at 15° C. for 12 hour. LC-MS showed DM1-SPP was consumed completely and one main peak with desired MS was detected. The DCM was removed and the residue was purified by prep-HPLC (neutral condition). Compound DM1-SPP-TFP (0.123 g, 120.76 μmol, 60.07% yield) was obtained as a white solid.

LCMS (ESI): 999.9 [M + H—H₂O]⁺ Molecular weight 1018.53

General Procedure for Coupling DM1-SPP-TFP with Targeting Bicycles

To a solution of targeting Bicycle (1.1-1.3 eq) in DMA was added DIEA (3 eq) and DM1-SPP-TFP (1 eq). The mixture was stirred at 25° C. for 16 hr. The reaction was monitored by LC-MS and once complete, the mixture was directly purified by preparative HPLC.

To a solution of DM1-SPP (30.00 mg, 34.46 μmol, 1.00 eq) in DMF (5.00 mL) was added DIEA (13.36 mg, 103.38 μmol, 18.0 μL, 3.00 eq) and HATU (13.10 mg, 34.46 μmol, 1.00 eq). After 1 h, BCY6014 (104.79 mg, 34.46 μmol, 1.00 eq) was added and the mixture was stirred at 15° C. for 2 hours. LC-MS showed 40% of DM1-SPP was remained. Several new peaks were observed on LC-MS and 20% of desired compound was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6032 (10.00 mg, 2.57 μmol, 7.45% yield) was obtained as a white solid.

BCY6032 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 25-55% B over 20 minutes, then 3 min 95% B Retention Time: 13.38 min LCMS (ESI): m/z 1292.1 [M + 3H— H2O]³⁺, 969.0 [M + 4H—H2O]⁴⁺ Peptide mw 3892.94

BCY6018 (86.96 mg, 32.40 μmol, 1.1 eq) was used as the bicycle reagent. BCY6052 (32.30 mg, 9.13 μmol, 31.01% yield) was obtained as a white solid.

BCY6052 Analytical Data Mobile Phase: A: 0.1% Formic acid in H2O B: ACN Flow: 1.0 ml/min Column: Eclipse XDB-Phenyl 3.5 um 100 * 3.0 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 6.96 min LCMS (ESI): m/z 1173.4 [M + 3H—H2O]³⁺, 884.6 [M + 4H]⁴⁺ Peptide mw 3536.58

BCY6017 (66.50 mg, 29.45 μmol, 1.2 eq) was used as the bicycle reagent. BCY6048 (40.80 mg, 13.12 μmol, 53.45% yield) was obtained as a white solid.

BCY6048 Analytical Data Mobile Phase: A: 0.1% Formic acid in H2O B: ACN Flow: 1.0 ml/min Column: Eclipse XDB-Phenyl 3.5 um 100 * 3.0 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 7.56 min LCMS (ESI): m/z 1031.0 [M + 3H—H2O]³⁺, 884.6 [M + 4H]⁴⁺ Peptide mw 3110.13

BCY6019 (113.60 mg, 32.40 μmol, 1.10 eq) was used as the bicycle reagent. BCY6036 (53.20 mg, 14.00 μmol, 47.54% yield, 96.26% purity) was obtained as a white solid.

BCY6036 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 8.19 min LCMS (ESI): m/z 1213.6 [M + 3H—H2O]³⁺, 914.7 [M + 4H]⁴⁺ Peptide mw 3657.76

BCY6009 (99.00 mg, 29.45 μmol, 1.00 eq) was used as the bicycle reagent. BCY6028 (24.30 mg, 6.05 μmol, 20.56% yield, 96.61% purity) was obtained as a white solid.

BCY6028 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 35-65% B over 20 minutes, then 3 min 95% B Retention Time: 6.43 min LCMS (ESI): m/z 965.6 [M + 4H—H2O]⁴⁺ Peptide mw 3877.96

To a solution of 2-(2-pyridyldisulfanyl)pyridine (2.46 g, 11.18 mmol, 1.50 eq) and AcOH (1.05 g, 17.49 mmol, 1.00 mL, 2.35 eq) in EtOH (50.00 mL) was added 4-sulfanylpentanoic acid (1A) (1.00 g, 7.45 mmol, 1.00 eq). The mixture was stirred at 40° C. for 18 hours under N₂. LC-MS showed 1A was consumed completely and one main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by preparative HPLC (neutral condition). Compound 2A (1.61 g, 6.62 mmol, 88.81% yield) was obtained as a light yellow solid.

LCMS (ESI): 243.9 [M + H]⁺ Molecular weight 243.34

To a solution of 2A (0.01 g, 41.09 μmol, 1.00 eq), 1-hydroxypyrrolidine-2,5-dione (14.19 mg, 123.28 μmol, 3.00 eq) in DMA (1 mL) was added EDCl (23.63 mg, 123.28 μmol, 3.00 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed 2A was consumed completely and one main peak with desired mass was detected. The residue was purified by preparative HPLC (neutral condition). Compound 3A (0.011 g, 32.31 μmol, 78.63% yield) was obtained as a white solid.

LCMS (ESI): 340.8 [M + H]⁺ Molecular weight 340.41

To a solution of BCY6014 (98.25 mg, 32.31 μmol, 1.00 eq) in DMA (3 mL) was added DIEA (8.26 mg, 64.62 μmol, 11.26 μL, 2.00 eq) and 3A (0.011 g, 32.31 μmol, 1.00 eq). The mixture was stirred at 15° C. for 18 hr. LC-MS showed 3A was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (neutral condition). Compound 4A (0.04 g, 12.25 μmol, 37.90% yield) was obtained as a white solid.

LCMS (ESI): 1088.7 [M + 3H]³⁺, 816.5 [M + 4H]⁴⁺ Molecular weight 3264.88

To a solution of 4A (0.04 g, 12.25 μmol, 1.00 eq) in MeCN (4 mL) and H₂O (2 mL) was added TCEP (4.21 mg, 14.70 μmol, 4.05 μL, 1.20 eq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed 4A was consumed completely and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (neutral condition). Compound 5A (0.035 g, 11.09 μmol, 90.53% yield) was obtained as a white solid.

LCMS (ESI): 1052.2 [M + 3H]³⁺ Molecular weight 3155.73

To a solution of 4-(2-pyridyldisulfanyl)pentanoic acid (2A) (22.46 mg, 92.29 μmol, 1.20 eq), HATU (35.09 mg, 92.29 μmol, 1.20 eq), DIEA (29.82 mg, 230.71 μmol, 40.19 μL, 3.00 eq) in DMF (5 mL) was added 1B (0.05 g, 76.90 μmol, 1.00 eq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed 1B was consumed completely and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (neutral condition). Compound DM3-SPy (0.025 g, 28.56 μmol, 37.13% yield) was obtained as a white solid.

LCMS (ESI): 875.1 [M + H]⁺ Molecular weight 875.49

A solution of DM3-SPy (0.015 g, 17.13 μmol, 1.00 eq) and 5A (54.08 mg, 17.13 μmol, 1.00 eq) in DMF (3 mL) was adjusted to pH=8 using NaHCO₃(aq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed DM3-SPy was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6039 (0.0263 g, 6.58 μmol, 38.39% yield) was obtained as a white solid.

BCY6039 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE (1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 13.01 min LCMS (ESI): m/z 976.1 [M + 4H—H2O]⁴⁺ Peptide mw 3921.01

To a solution of compound 1 (0.045 g, 126.96 μmol, 1 eq) in H₂O (1 mL) was adjusted pH=13 using 1 N NaOH solution. The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 1 was consumed completely and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (neutral condition). Compound 2 (0.03 g, 116.56 μmol, 91.81% yield) was obtained as a yellow solid.

LCMS (ESI): 257.9 [M + H]⁺ Molecular weight 257.37

A solution of compound 2 (0.03, 116.56 μmol, 1.0 eq) and DM1 (111.87 mg, 151.53 μmol, 1.3 eq) in DMF (5 mL) was stirred at 15° C. for 2 hours. LC-MS showed DM1 was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (NH₄HCO₃ condition). Compound 3 (0.05 g, 56.53 μmol, 48.50% yield) was obtained as a white solid.

LCMS (ESI): 866.0 [M + H—H₂O]⁺ Molecular weight 884.49

To a solution of compound 3 (0.05 g, 56.53 μmol, 1.0 eq) and 2,3,5,6-tetrafluorophenol (28.16 mg, 169.59 μmol, 3.0 eq) in DMA (3 mL) and DCM (1 mL) was added EDCl (32.51 mg, 169.59 μmol, 3 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired mass was detected. DCM was removed and the mixture was directly purified by preparative HPLC (neutral condition). Compound 4 (0.03 g, 29.05 μmol, 51.40% yield) was obtained as a white solid.

LCMS (ESI): 1014.0 [M + H—H₂O]⁺ Molecular weight 1032.55

To a solution of BCY6014 (106.01 mg, 34.87 μmol, 1.2 eq) in DMA (3 mL) was added DIEA (11.27 mg, 87.16 μmol, 15.18 μL, 3.0 eq) and compound 4 (0.03 g, 29.05 μmol, 1.0 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 4 was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6055 (0.0352 g, 9.01 μmol, 31.01% yield) was obtained as a white solid.

BCY6055 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE (1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 12.68 min LCMS (ESI): m/z 1296.1 [M + 3H—H2O]³⁺, 972.4 [M + 4H—H2O]⁴⁺ Peptide mw 3906.98

General Procedure for Preparation of Compound 2

To a solution of compound 1 (0.1 g, 410.94 μmol, 1 eq) in 1,2-dichloroethane (3 mL) was added sulfurochloridic acid (0.86 g, 7.38 mmol, 491.43 μL, 17.96 eq) on three parts and DIEA (318.67 mg, 2.47 mmol, 429.47 μL, 6 eq) was added on two parts. The mixture was stirred at 75° C. for 16 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected MS324, one main peak of byproduct MS 221 was PySSPy. The solvent was removed and dissolved in H2O/MeCN=15/1. Directly purified by prep-HPLC (neutral condition: MeCN/H₂O). Compound 2 (0.055 g, 170.06 μmol, 41.38% yield) was obtained as a yellow oil.

LCMS (ESI): 323.6 [M + H]⁺ Molecular weight 323.4

General Procedure for Preparation of DM1-SO3H-SPP

To a solution of DM1 (113.00 mg, 153.06 μmol, 1.1 eq), compound 2 (0.045 g, 139.14 μmol, 1 eq) in DMF (2 mL) was adjusted PH=8 used for NaHCO₃(aq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed DM1 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound DM1-SO3H-SPP (0.075 g, 78.90 μmol, 56.71% yield) was obtained as a white solid.

LCMS (ESI): 931.9 [M + H—H2O]⁺ Molecular weight 950.52

General Procedure for Preparation of DM1-SO3H-SPP-NHS

To a solution of DM1-SO3H-SPP (0.06 g, 63.12 μmol, 1 eq), 1-hydroxypyrrolidine-2, 5-dione (7.99 mg, 69.43 μmol, 1.1 eq) in DMA (1.5 mL) and DCM (0.5 mL) was added EDCl (13.31 mg, 69.43 μmol, 1.1 eq). The mixture was stirred at 15° C. for 18 hr. LC-MS showed DM1-SO3H-SPP was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition: MeCN/H₂O). Compound DM1-SO3H-SPP-NHS (0.045 g, 42.96 μmol, 68.05% yield) was obtained as a white solid.

LCMS (ESI): 984 [M − NHS + K]⁺ Molecular weight 1047.6

General Procedure for Preparation of BCY6077

To a solution of BCY6014 (101.58 mg, 33.41 μmol, 1 eq) in DMA (1 mL) was added DIEA (12.95 mg, 100.23 μmol, 17.46 μL, 3 eq) and DM1-SO3H-SPP-TFP (0.035 g, 33.41 μmol, 1 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed DM1-SO3H-SPP-TFP was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (TFA condition). Compound BCY6077 (41.30 mg, 10.03 μmol, 30.01% yield, 96.44% purity) was obtained as a white solid.

BCY6077 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE (1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 11.80 min LCMS (ESI): m/z 978.2 [M + 4H − 18 − 44]⁴⁺ Peptide mw 3972.06

Non-Cleavable Series

To a solution of MMAE (0.2 g, 278.56 μmol, 1.0 eq) in DMA (3 mL) was added DIEA (108.01 mg, 835.68 μmol, 145.56 μL, 3.0 eq) and tetrahydropyran-2,6-dione (63.57 mg, 557.12 μmol, 2.0 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed MMAE was consumed completely and one main peak with desired mass was detected. The mixture was The mixture was directly purified by preparative HPLC (neutral condition). Compound Glutarate-MMAE (0.12 g 144.22 μmol, 51.77% yield) was obtained as a white solid.

LCMS (ESI): 832.3 [M + H]+ Molecular weight 832.09

To a solution of Glutarate-MMAE (0.12 g, 144.22 μmol, 1.0 eq), 1-hydroxypyrrolidine-2, 5-dione (49.79 mg, 432.65 μmol, 3.0 eq) in DMA (3 mL) and DCM (1 mL) was added EDCl (82.94 mg, 432.65 μmol, 3.0 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed Glutarate-MMAE was consumed completely and one main peak with desired mass was detected. The mixture was The mixture was directly purified by preparative HPLC (TFA condition). Compound Glutarate-MMAE-NHS (0.055 g, 59.19 μmol, 41.04% yield) was obtained as a white solid.

LCMS (ESI): 929.2 [M + H]⁺ Molecular weight 929.17

To a solution of BCY6014 (98.17 mg, 32.29 μmol, 1.2 eq) in DMA (2 mL) were added DIEA (10.43 mg, 80.72 μmol, 14.06 μL, 3 eq) and Glutarate-MMAE-NHS (0.025 g, 26.91 μmol, 1 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed Glutarate-MMAE-NHS was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6063 (32.10 mg, 8.33 μmol, 30.95% yield) was obtained as a white solid.

BCY6063 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 10.86 min LCMS (ESI): m/z 963.8 [M + 4H]⁴⁺, 771.1 [M + 5H]⁵⁺ Peptide mw 3854.56

To a solution of DM1 (0.1 g, 135.45 μmol, 1 eq), 3-[(2-bromoacetyl)amino]propanoic acid (34.14 mg, 162.54 μmol, 1.2 eq) in DMF (5 mL) was added TEA (41.12 mg, 406.35 μmol, 56.56 μL, 3 eq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed DM1 was consumed completely and one main peak with the desired mass was detected. The mixture was directly purified by preparative HPLC (neutral conditions). Compound 1 (0.08 g, 92.23 μmol, 68.09% yield) was obtained as a white solid.

LCMS (ESI): 849.1 [M + H—H₂O]⁺ Molecular weight 867.41

To a solution of compound 1 (0.08 g, 92.23 μmol, 1 eq), 2,3,5,6-tetrafluorophenol (45.95 mg, 276.69 μmol, 3 eq) in DMA (3 mL) and DCM (1 mL) was added EDCl (53.04 mg, 276.69 μmol, 3 eq). The mixture was stirred at 15° C. for 4 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (neutral condition). Compound 2 (0.06 g, 59.09 μmol, 64.06% yield) was obtained as a white solid.

LCMS (ESI): 997.0 [M + H—H₂O]⁺ Molecular weight 1015.46

To a solution of BCY6014 (107.79 mg, 35.45 μmol, 1.2 eq) in DMA (3 mL) was added DIEA (11.45 mg, 88.63 μmol, 15.44 μL, 3.0 eq) and compound 2 (0.030 g, 29.54 μmol, 1 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6064 (28.40 mg, 7.30 μmol, 24.71% yield) was obtained as a white solid.

BCY6064 Analytical Data Mobile Phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 um 110A 150 * 4.6 mm Instrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30 minutes, then 3 min 95% B Retention Time: 10.26 min LCMS (ESI): m/z 968.4 [M + 4H—H2O]⁴⁺ Peptide mw 3889.89

General Procedure for Preparation of Compound 2

To a solution of compound 1 (3.5 g, 5.68 mmol, 1.0 eq) in DCM (20 mL) and MeOH (10 mL), (4-aminophenyl)methanol (978.5 mg, 7.95 mmol, 1.4 eq) and EEDQ (2.81 g, 11.35 mmol, 2.0 eq) were added in the dark, and the mixture was stirred at 25° C. for 18 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected ([M+H⁺]=722.0). The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash®Silica Flash Column, Eluent of 0-10% Methanol/Dichloromethane @ 80 mL/min). Compound 2 (3.0 g, 4.16 mmol, 73.2% yield) was obtained as a yellow solid.

General Procedure for Preparation of Compound 3

To a solution of compound 2 (2.5 g, 3.46 mmol, 1.0 eq) in THF (30 mL) was added DIEA (2.69 g, 20.78 mmol, 3.62 mL, 6.0 eq) and bis(4-nitrophenyl) carbonate (6.32 g, 20.78 mmol, 6.0 eq), and the mixture was stirred at 25° C. for 16 hr. TLC indicated compound 2 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0-5% Methanol/Dichloromethane @ 100 mL/min). Compound 3 (2.2 g, 2.48 mmol, 71.6% yield) was obtained as a yellow solid.

General Procedure for Preparation of Compound 4

To a solution of compound 3 (0.3 g, 338.24 umol, 1.0 eq) in DMF (5 mL), HOBt (50.3 mg, 372.06 umol, 1.1 eq), DIEA (131.1 mg, 1.01 mmol, 176.7 μL, 3.0 eq), and MMAE (218.6 mg, 304.42 umol, 0.9 eq) were added. The mixture was stirred at 40° C. for 16 hr. LC-MS showed one peak with desired MS ([M+H⁺]=1466.4, [M+2H⁺]/2=733.2). The reaction mixture was then directly purified by prep-HPLC (neutral condition), and compound 4 (0.2 g, 136.44 umol, 40.3% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 5

Compound 4 (0.175 g, 119.39 umol, 1.0 eq) was first dissolved in TFA (1.8 mL), and then triisopropylsilane (13.5 g, 85.20 mmol, 17.5 mL, 713.7 eq) was added. The mixture was stirred at 0° C. for 30 min. LC-MS showed one peak with desired MS ([M+H⁺]=1123.4, [M+2H⁺]/2=562.2). The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 5 (0.1 g, 89.02 umol, 74.6% yield) was obtained as a yellow solid.

General Procedure for Preparation of Compound 6

To a solution of compound 5 (0.07 g, 62.31 umol, 1.0 eq) in DMA (1.0 mL), DIEA (24.2 mg, 186.94 umol, 32.6 μL, 3.0 eq) and tetrahydropyran-2,6-dione (14.2 mg, 124.62 umol, 2.0 eq) were added. The mixture was stirred at 25° C. for 2 hr. LC-MS showed compound 5 was consumed completely and one main peak with desired MS was detected ([M+H⁺]=1237.4, [M+2H⁺]/2=619.3). The reaction mixture was then directly purified by prep-HPLC (neutral condition), and compound 6 (0.04 g, 32.32 umol, 51.8% yield) was obtained as a light yellow solid.

General Procedure for Preparation of Compound 7

To a solution of compound 6 (0.04 g, 32.32 umol, 1.0 eq), 1-hydroxypyrrolidine-2,5-dione (11.2 mg, 96.97 umol, 3.0 eq) in DMA (3.0 mL) and DCM (1.0 mL), EDCl (18.6 mg, 96.97 umol, 3.0 eq) was added. The mixture was stirred at 25° C. for 18 hr. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected ([M+H⁺]=1334.5, [M+2H⁺]/2=667.7). The reaction mixture was then directly purified by prep-HPLC (TFA condition), and compound 7 (0.025 g, 18.73 umol, 57.9% yield) was obtained as a white solid.

General Procedure for Preparation of BCY6105

To a solution of BCY6014 (82.0 mg, 26.98 umol, 1.2 eq) in DMA (4 mL), DIEA (8.7 mg, 67.44 umol, 11.7 μL, 3.0 eq) and compound 7 (0.03 g, 22.48 umol, 1.0 eq) were added. The mixture was stirred at 25° C. for 18 hr. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected ([M+4H⁺]/4=1065.2). The reaction mixture was then directly purified by prep-HPLC (TFA condition). Compound BCY6105 (0.024 g, 5.41 umol, 24.1% yield, 96.06% purity) was obtained as a white solid.

General Procedure for Preparation of Compound 2

To a solution of compound 1 (5.0 g, 10.67 mmol, 1.0 eq) in DCM (30 mL) and MeOH (10 mL), EEDQ (5.28 g, 21.34 mmol, 2.0 eq) and (4-aminophenyl)methanol (2.63 g, 21.34 mmol, 2.0 eq) were added. The mixture was stirred at 20° C. for 18 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected (desired m/z=574, while Boc group falling off and partially falling off corresponded to m/z=474 and 518, respectively). The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 2 (3.7 g, 6.45 mmol, 60.4% yield) was obtained as a yellow solid.

General Procedure for Preparation of Compound 3

To a solution of compound 2 (3.4 g, 5.93 mmol, 1.0 eq) in DMF (20 mL) was added DIEA (5.36 g, 41.49 mmol, 7.23 mL, 7.0 eq) and bis(4-nitrophenyl) carbonate (10.82 g, 35.56 mmol, 6.0 eq) in one part. The mixture was stirred at 25° C. for 2 hr. LC-MS showed one peak with desired MS (m/z=639 corresponded to the mass with Boc group falling off during ESI). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 3 (3.0 g, 4.06 mmol, 68.5% yield) was obtained as a yellow solid.

General Procedure for Preparation of Compound 4

To a solution of compound 3 (707.4 mg, 957.55 umol, 1.0 eq) in DMF (15 mL), HOBt (155.3 mg, 1.15 mmol, 1.2 eq), DIEA (371.3 mg, 2.87 mmol, 500.4 μL, 3.0 eq), and MMAE (0.55 g, 766.04 umol, 0.8 eq) were added. The mixture was stirred at 30° C. for 16 hr. LC-MS showed one peak with desired MS (desired m/z=1317, and m/z=609 corresponded to the mass with two protons and Boc group falling off during ESI). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 4 (0.53 g, 402.23 umol, 42.0% yield) was obtained as a yellow solid.

General Procedure for Preparation of Compound 5

To a solution of compound 4 (0.526 g, 399.20 umol, 1.0 eq) in DMF (4 mL), piperidine (862.2 mg, 10.13 mmol, 1.0 mL, 25.4 eq) was added. The mixture was stirred at 25° C. for 30 min. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected (desired m/z=1095, and m/z=265 corresponded to Fmoc-piperidine adduct). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 5 (0.230 g, 209.97 umol, 52.6% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 6

To a solution of Fmoc-(D-Ala)-Phe-OH (125.6 mg, 273.87 umol, 1.2 eq) in DMF (10 mL), EDCl (52.5 mg, 273.87 umol, 1.2 eq), HOBt (37.0 mg, 273.87 umol, 1.2 eq), and compound 5 (0.25 g, 228.23 umol, 1 eq) were added. The mixture was stirred at 25° C. for 3 hr. LC-MS showed compound 5 was consumed completely and one peak with desired MS was detected (m/z=718 corresponded to the mass with two protons and Boc group falling off during ESI). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 6 (0.18 g, 117.20 umol, 51.3% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 7

To a solution of compound 6 (0.18 g, 117.20 umol, 1.0 eq) in DMF (8 mL), piperidine (1.72 g, 20.25 mmol, 2.0 mL, 172.8 eq) was added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected (m/z=1314 and 657 corresponded to the desired mass, and m/z=265 corresponded to Fmoc-piperidine adduct). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 7 (0.13 g, 98.96 umol, 84.4% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 8

To a solution of compound 7 (0.105 g, 79.93 umol, 1.0 eq) in DMA (4 mL), DIEA (31.0 mg, 239.79 umol, 41.8 μL, 3.0 eq) and tetrahydropyran-2,6-dione (27.4 mg, 239.79 umol, 3.0 eq) were added. The mixture was stirred at 25° C. for 2 hr. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected (m/z 664.5 corresponded to the mass with two protons and Boc group falling off during ESI). The reaction mixture was then directly purified by prep-HPLC (neutral condition), and compound 8 (0.09 g, 63.04 umol, 78.8% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 9

To a solution of compound 8 (0.09 g, 63.04 umol, 1.0 eq), 1-hydroxypyrrolidine-2,5-dione (21.7 mg, 189.11 umol, 3.0 eq) in DMA (3 mL) and DCM (1 mL), EDCl (36.2 mg, 189.11 umol, 3.0 eq) dissolved in 1 mL DCM was added. The mixture was stirred at 25° C. for 18 hr. LC-MS showed compound 8 was consumed completely and one main peak with desired MS was detected (desired m/z=1524 (one proton) and 763 (two protons), while m/z=713 corresponded to the mass with Boc group falling off during ESI). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 9 (0.07 g, 45.91 umol, 72.8% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 10

To a solution of BCY6014 (167.5 mg, 55.09 umol, 1.2 eq) in DMF (3 mL), DIEA (11.8 mg, 91.81 umol, 16.0 μL, 2.0 eq) and compound 9 (0.07 g, 45.91 umol, 1.0 eq) were added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 9 was consumed completely and one main peak with desired MS was detected ([M+4H⁺]/4=1112.9, [M+5H⁺]/5=890.5). The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The crude product 10 (0.220 g, crude) was used in the next step without further purification.

General Procedure for Preparation of BCY6106

To a solution of compound 10 (0.200 g, 44.95 umol, 1.0 eq) in DCM (4 mL), 1 mL TFA was added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed one main peak with desired MS ([M+4H⁺]/4=1088.0, [M+5H⁺]/5=870.8). The reaction mixture was concentrated under reduced pressure to give a residue, which was then directly purified by prep-HPLC (TFA condition). Compound BCY6106 (0.0297 g, 20.06 umol, 14.5% yield, 95.46% purity) was obtained as a white solid.

General Procedure for Preparation of Compound 9

The synthesis of Compound 9 was performed in an analogous to manner to that described in BCY6106.

General Procedure for Preparation of Compound 10A

To a solution of BCY6099 (195.15 mg, 61.32 μmol, 1.1 eq) in DMA (3 mL) were added DIEA (21.61 mg, 167.23 μmol, 29.13 μL, 3 eq) and compound 9 (0.085 g, 55.74 μmol, 1.0 eq). The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 9 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to afford a residue (light yellow oil). The reaction was directly purified by prep-HPLC (neutral condition). Compound 10A (0.160 g, 34.84 μmol, 62.50% yield) was obtained as a white solid.

General Procedure for Preparation of BCY6175

To a solution of compound 10A in DCM (4.5 mL) was added TFA (4.5 mL). The mixture was stirred at 0° C. for 30 min. LC-MS showed compound 10A was consumed completely and one main peak with desired m/z was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to afford a residue, which was purified by prep-HPLC (TFA condition). Compound BCY6175 (61.40 mg, 13.56 μmol, 31.13% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 2

To a solution of compound 1 (3.0 g, 8.49 mmol, 1.0 eq) in DCM (30 mL) and MeOH (10 mL), EEDQ (2.52 g, 10.19 mmol, 1.2 eq) and (4-aminophenyl)methanol (1.25 g, 10.19 mmol, 1.2 eq) were added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected ([M+H]⁺459.5). In addition, TLC indicated compound 1 was consumed completely and new spots formed. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-60% Ethylacetate/Petroleum ether gradient @ 80 mL/min). Compound 2 (3.5 g, 7.63 mmol, 89.9% yield) was obtained as a yellow solid.

General Procedure for Preparation of Compound 3

To a solution of compound 2 (3.3 g, 7.20 mmol, 1.0 eq) in THF (100 mL), DIEA (4.65 g, 35.98 mmol, 6.27 mL, 5.0 eq) and bis(4-nitrophenyl) carbonate (8.76 g, 28.79 mmol, 4.0 eq) were added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired MS was detected ([M+H]⁺624.0). In addition, TLC indicated compound 2 was consumed completely and new spots formed. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-15% Ethylacetate/Petroleum ether gradient @80 mL/min). Compound 3 (3.0 g, 4.81 mmol, 66.8% yield) was obtained as a yellow solid.

General Procedure for Preparation of Compound 4

To a solution of compound 3 (124.09 mg, 198.97 umol, 1.0 eq) in DMF (5 mL), HOBt (32.3 mg, 238.77 umol, 1.2 eq), DIEA (77.1 mg, 596.92 μmol, 103.9 μL, 3.0 eq), and MMAE (0.1 g, 139.28 umol, 0.7 eq) were added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired MS was detected ([M+H]⁺1202.5, [M+Na]⁺1224.5). The reaction mixture was then directly purified by prep-HPLC (neutral condition). After lyophilization, compound 4 (0.08 g, 66.53 umol, 33.4% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 5

To a solution of compound 4 (0.08 g, 66.53 umol, 1.0 eq) in DMF (4 mL), piperidine (862.2 mg, 10.13 mmol, 1 mL, 152.2 eq) was added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected ([M+H]⁺981.5, [M+Na]⁺1003.5, while m/z=264.0 corresponded to Fmoc-piperidine adduct). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 5 (0.055 g, 56.11 umol, 84.3% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 6

To a solution of Fmoc-Glu(t-Bu)-Pro-Cit-Gly-HPhe-Tyr(t-Bu)-OH (74.1 mg, 66.31 umol, 1.3 eq) in DMF (4 mL), EDCl (12.7 mg, 66.31 umol, 1.3 eq), HOBt (8.9 mg, 66.31 umol, 1.3 eq), and compound 5 (0.05 g, 51.01 umol, 1.0 eq) were added. The mixture was stirred at 25° C. for 30 min. LC-MS indicated 20% of compound 5 remained, several new peaks formed, and 60% of the reaction mixture was desired product ([M+2H⁺]/2=1040.4). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 6 (0.07 g, 33.66 umol, 66.0% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 7

To a solution of compound 6 (0.07 g, 33.66 umol, 1.0 eq) in DMF (4 mL), piperidine (2.9 mg, 33.66 umol, 3.3 μL, 1.0 eq) was added. The mixture was stirred at 25° C. for 15 min. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected ([M+2H⁺]/2=929.1, while m/z=264.2 corresponded to Fmoc-piperidine adduct). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 7 (0.045 g, 24.23 umol, 72.0% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 8

To a solution of compound 7 (0.04 g, 21.54 umol, 1.0 eq) in DMA (1 mL), DIEA (8.3 mg, 64.61 umol, 11.2 μL, 3.0 eq) and tetrahydropyran-2,6-dione (7.4 mg, 64.61 umol, 3.0 eq) were added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected ([M+2H⁺]/2=986.4). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 8 (0.035 g, 17.75 umol, 82.4% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 9

To a solution of compound 8 (0.035 g, 17.75 umol, 1.0 eq), 1-hydroxypyrrolidine-2,5-dione (6.1 mg, 53.26 umol, 3.0 eq) in DMA (3 mL) and DCM (1 mL), EDCl (10.2 mg, 53.26 umol, 3.0 eq) was added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 8 was partially remained and one peak with desired MS was detected ([M+2H⁺]/2=1034.7). DCM was then removed, following by mixture being purified by prep-HPLC (neutral condition). Compound 9 (0.03 g, 14.50 umol, 81.7% yield) was obtained as a white solid.

General Procedure for Preparation of Compound 10

To a solution of BCY6014 (52.9 mg, 17.40 umol, 1.59 μL, 1.2 eq) in DMF (2 mL), DIEA (5.6 mg, 43.51 umol, 7.6 μL, 3.0 eq) and compound 9 (0.03 g, 14.50 umol, 1.0 eq) were added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed one main peak with desired MS ([M+4H⁺]/4=1249.2, [M+5H⁺]/5=999.3). The solvent was then removed and the resulting crude product 10 (0.06 g, crude) was used into the next step without further purification.

General Procedure for Preparation of BCY6107

To a solution of compound 10 (0.055 g, 11.01 umol, 1.0 eq) in DCM (1 mL), 1 mL TFA was added. The mixture was stirred at 0° C. for 15 min. LC-MS showed compound 10 was consumed completely and one main peak with desired MS was detected ([M+4H⁺]/4=1221.0, [M+5H⁺]/5=977.0). The reaction mixture was concentrated under reduced pressure to remove solvent, resulting a residue which was then directly purified by prep-HPLC (TFA condition). Compound BCY6107 (20.4 mg, 4.03 umol, 36.6% yield, 96.36% purity) was obtained as a white solid.

Biological Data

Study 1: Fluorescence Polarisation Measurements

(a) Direct Binding Assay

Peptides with a fluorescent tag (either fluorescein, SIGMA or Alexa Fluor488™, Fisher Scientific) were diluted to 2.5 nM in PBS with 0.01% tween 20 or 50 mM HEPES with 100 mM NaCl and 0.01% tween pH 7.4 (both referred to as assay buffer). This was combined with a titration of protein in the same assay buffer as the peptide to give 1 nM peptide in a total volume of 25 μL in a black walled and bottomed low bind low volume 384 well plates, typically 5 μL assay buffer, 10 μL protein (Table 1) then 10 μL fluorescent peptide. One in two serial dilutions were used to give 12 different concentrations with top concentrations ranging from 500 nM for known high affinity binders to 10 μM for low affinity binders and selectivity assays. Measurements were conducted on a BMG PHERAstar FS equipped with an “FP 485 520 520” optic module which excites at 485 nm and detects parallel and perpendicular emission at 520 nm. The PHERAstar FS was set at 25° C. with 200 flashes per well and a positioning delay of 0.1 second, with each well measured at 5 to 10 minute intervals for 60 minutes. The gain used for analysis was determined for each tracer at the end of the 60 minutes where there was no protein in the well. Data was analysed using Systat Sigmaplot version 12.0. mP values were fit to a user defined quadratic equation to generate a Kd value: f=ymin+(ymax-ymin)/Lig*((x+Lig+Kd)/2−sqrt((((x+Lig+Kd)/2){circumflex over ( )}2)−(Lig*x))). “Lig” was a defined value of the concentration of tracer used.

(b) Competition Binding Assay

Peptides without a fluorescent tag were tested in competition with a peptide with a fluorescent tag and a known Kd (Table 2). Reference Compound A has the sequence FI-G-Sars-ACPWGPAWCPVNRPGCA (FI-G-Sars-(SEQ ID NO: 94)). Reference Compound B has the sequence FI-G-Sars-ACPWGPFWCPVNRPGCA (FI-G-Sars-(SEQ ID NO: 95)). Reference Compound C has the sequence FI-G-Sars-ADVTCPWGPFWCPVNRPGCA (FI-G-Sars-(SEQ ID NO: 96). Each of Reference Compounds A, B and C contain a TBMB molecular scaffold. Peptides were diluted to an appropriate concentration in assay buffer as described in the direct binding assay with a maximum of 5% DMSO, then serially diluted 1 in 2. Five μL of diluted peptide was added to the plate followed by 10 μL of human or mouse EphA2 (Table 1) at a fixed concentration which was dependent on the fluorescent peptide used (Table 2), then 10 μL fluorescent peptide added. Measurements were conducted as for the direct binding assay, however the gain was determined prior to the first measurement. Data analysis was in Systat Sigmaplot version 12.0 where the mP values were fit to a user defined cubic equation to generate a Ki value:

f=ymin+(ymaxdymin)/Lig*((Lig*((2*((Kig+Kcomp+Lig+CompSProt*c){circumflex over ( )}2−3*(Kcomp*(Ligb Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}3){circumflex over ( )}0.5)))/3))>(Klig+Kcomp+Lig+Comp−Prot*c)))/((3*Klig)+((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Kig*Kcomp)){circumflex over ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+KIig*(Comp−Prot*c)+KIig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−. Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}3){circumflex over ( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))). “Lig”, “KLig” and “Prot” were all defined values relating to: fluorescent peptide concentration, the Kd of the fluorescent peptide and EphA2 concentration respectively.

TABLE 1 Ephrin receptors and source Receptor Catalogue (domain) Species Format/tag Supplier number EphA1 (Ecto) Human Fc fusion R&D systems 7146-A1 EphA2 (Ecto) Human C-terminal R&D systems 3035-A2 polyHis EphA2 (Ecto) Human C- In-house N/A terminal polyHis EphA2 (Ecto) Mouse Fc fusion R&D Systems  639-A2 EphA2 (Ecto) Mouse C- Sino 50586- terminal polyHis Biological M08H EphA2 (ligand Rat C- In-house N/A binding) terminal polyHis EphA2 (ligand Dog C- In-house N/A binding) terminal polyHis EphA3 (Ecto) Human Fc fusion R&D systems 6444-A3 EphA3 (Ecto) Human N- In-house N/A terminal polyHis EphA3 (Ecto) Rat C- Sino 80465- terminal polyHis Biological R08H EphA4 (Ecto) Human Fc fusion R&D systems 6827-A4 EphA4 (Ecto) Human C- Sino 11314- terminal polyHis Biological H08H EphA4 (Ecto) Rat C- Sino 80123- terminal polyHis Biological R08H EphA6 (Ecto) Human Fc fusion R&D systems 5606-A6 EphA7 (Ecto) Human Fc fusion R&D systems 6756-A7 EphB1 (Ecto) Rat Fc fusion R&D systems 1596-B1 EphB4 (Ecto) human C-terminal R&D systems 3038-B4 polyHis

TABLE 2 Final concentrations of fluorescent peptide and EphA2 as used with Competition Binding Assays Concentration of Concentration of Concentration of Fluorescent fluorescent peptide Human EphA2 Mouse EphA2 peptide (nM) (nM) (nM) Reference 10 75 Compound A Reference 1 30 Compound B Reference 0.8 (human) 1 (mouse) 2.4 50 Compound C

Certain peptide ligands of the invention were tested in the above mentioned assays and the results are shown in Tables 3-7:

TABLE 3  Biological Assay Data for Peptide Ligands of the Invention (TATA peptides, Direct Binding Assay) Bicycle Compound Human EphA2 (K_(D), Number Sequence nM ± 95% CI) 1 ACMNDWWCAMGWKCA-Sar₆-K(FI) ((SEQ ID NO: 3)-Sar₆-K(FI))   304 ± 91.99 2 ACVPDRRCAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 4)-Sar₆-K(FI)) 74.91 ± 6.6   3 ACVVDGRCAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 5)-Sar₆-K(FI)) 129.8 ± 80.75 4 ACVVDSRCAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 6)-Sar₆-K(FI)) 124.6 ± 51.74 5 ACVPDSRCAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 7)-Sar₆-K(FI)) 93.95 ± 23.62 6 ACYVGKECAIRNVCA-Sar₆-K(FI) ((SEQ ID NO: 8)-Sar₆-K(FI)) 168.5 ± 20.58 7 ACYVGKECAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 9)-Sar₆-K(FI)) 149.73 ± 39.2   8 FI-G-Sar₅-ACYVGKECAYMNVCA (FI-G-Sar₅-(SEQ ID NO: 9)) 218.33 ± 10.51  9 FI-(β-Ala)-Sar₁₀-ARDCPLVNPLCLHPGWTC (FI-(β-Ala)-Sar₁₀-(SEQ ID NO: 10)) 6.43 ± 1.15 10 FI-(β-Ala)-Sar₁₀-A(HArg)DCPLVNPLCLHPGWTC (FI-(β-Ala)-Sar₁₀-(SEQ ID NO: 11) 9.07 ± 2.49 11 Ac-CPLVNPLCLHPGWTCLHG-Sar₆-(D-K[FI]) (Ac-(SEQ ID NO: 12)-Sar₆-(D-K[FI])) 3.08 ± 0.43 12 Ac-CPLVNPLCLHPGWTCL(D-His)G-Sar₆-(D-K[FI]) (Ac-SEQ ID NO: 13)-Sar₆-(D-K[FI])) 10.56 ± 0.77  13 Ac-CPLVNPLCLHPGWSCRGQ-Sar₆-(D-K[FI]) (Ac-(SEQ ID NO: 14)-Sar₆-(D-K[FI])) 5.29 ± 0.79 14 Ac-CPLVNPLCLHPGWSC(HArg)GQ-Sar₆-(D-K[FI]) (Ac-(SEQ ID NO: 15)-Sar₆-(D-K[FI])) 9.96 ± 0.55

TABLE 4  Biological Assay Data for Peptide Ligands of the Invention (TATA peptides, Competition Binding Assay) Ki, nM ± 95% CI Human EphA2 Mouse EphA2 Bicycle Fluorescent Peptide Compound Reference Reference Reference Reference Number Sequence Compound C Compound B Compound A Compound C 15 ACMNDWWCAMGWKCA 277.5 ± 38.22 (SEQ ID NO: 3) 16 ACVPDRRCAYMNVCA 69.97 ± 8.67  (SEQ ID NO: 4) 17 (β-Ala)-Sar₁₀- 85.05 ± 1.08  ACVPDRRCAYMNVC ((β-Ala)-Sar₁₀- (SEQ ID NO: 16)) 18 DLRCGGDPRCAYMNVCA 70.8 ± 2.35 (SEQ ID NO: 17) 19 SRPCVIDSRCAYMNVCA 94.75 ± 24.01 (SEQ ID NO: 18) 20 ESRCSPDARCAYMNVCA 57.05 ± 4.61  (SEQ ID NO: 19) 21 HSGCRPDPRCAYMNVCA 62.15 ± 4.61  (SEQ ID NO: 20) 22 GSGCKPDSRCAYMNVCA 63.25 ± 13.82 (SEQ ID NO: 21) 23 ETVCLPDSRCAYMNVCA   130 ± 15.68 (SEQ ID NO: 22) 24 GQVCIVDARCAYMNVCA 168.5 ± 16.66 (SEQ ID NO: 23) 25 ACVPDRRCAFENVCVDH 97.3 ± 3.33 (SEQ ID NO: 24) 26 ACVPDRRCAFMNVCEDR 39.05 ± 10.29 (SEQ ID NO: 25) 27 ACVPDRRCAFQDVCDHE 159 n = 1 (SEQ ID NO: 26) 28 ACVPDRRCAFRDVCLTG 1700 n = 1 (SEQ ID NO: 27) 29 ACYVGKECAYMNVCA  209.5 ± 110.74 106.65 ± 24.94 87.7 n = 1 (SEQ ID NO: 9) 30 ACQPSNHCAFMNYCA 293 n = 1 186.53 ± 86.86 137 n = 1 (SEQ ID NO: 28) 31 ACSPTPACAVQNLCA 223 n = 1    177 ± 60.76 (SEQ ID NO: 29) 32 ACTSCWAYPDSFCA   232 ± 52.19 151 n = 1 (SEQ ID NO: 30) 33 ACTKPTGFCAYPDTICA 268.5 ± 16.66 (SEQ ID NO: 31) 34 ACRGEWGYCAYPDTICA 347.5 ± 57.82 (SEQ ID NO: 32) 35 ACRNWGMYCAYPDTICA 282.5 ± 65.66 (SEQ ID NO: 33) 36 ACPDWGKYCAYPDTICA  160 ± 1.96 (SEQ ID NO: 34) 37 ACRVYGPYCAYPDTICA 294.5 ± 20.58 (SEQ ID NO: 35) 38 ACSSCWAYPDSVCA 400.33 ± 205.19 (SEQ ID NO: 36) 39 ACQSCWAYPDTYCA 321.33 ± 119.53 (SEQ ID NO: 37) 40 ACGFMGLEPCETFCA 187.5 ± 20.58 (SEQ ID NO: 38) 41 ACGFMGLVPCEVHCA 155 ± 9.8 (SEQ ID NO: 39) 42 ACGFMGLEPCEMVCA 320.5 ± 14.7  (SEQ ID NO: 40) 43 ACGFMGLEPCVTYCA 233.5 ± 20.58 (SEQ ID NO: 41) 44 ACGFMGLEPCELVCA 126.8 ± 21.17 (SEQ ID NO: 42) 45 ACGFMGLVPCNVFCA   142 ± 41.16 (SEQ ID NO: 43) 46 ACGFMGLEPCELFCA 81.7 ± 7.06 (SEQ ID NO: 44) 47 ACGFMGLEPCELFCMPK   185 ± 74.48 (SEQ ID NO: 45) 48 ACGFMGLEPCELYCA 127.5 ± 14.7  (SEQ ID NO: 46) 49 ACGFMGLEPCELYCAHT   144 ± 17.64 (SEQ ID NO: 47) 50 ACGFMGLEPCEMYCA   140 ± 45.08 (SEQ ID NO: 48) 51 ACGFMGLVPCELYCADN  84.4 ± 36.46 (SEQ ID NO: 49) 52 ACPLVNPLCLTSGWKCA 115.33 ± 11.33  (SEQ ID NO: 50) 53 ACPMVNPLCLHPGWICA 15.4 ± 3.17 (SEQ ID NO: 51) 54 ACPLVNPLCLHPGWICA 15.25 ± 2.84  (SEQ ID NO: 52) 55 ACPLVNPLCLHPGWRCA 20.55 ± 0.88  (SEQ ID NO: 53) 56 ACPLVNPLCNLPGWTCA    184 ± 115.64 (SEQ ID NO: 54) 57 ACPLVNPLCLVPGWSCA 35.4 ± 10   (SEQ ID NO: 55) 58 ACPLVNPLCLLDGWTCA 38.35 ± 5.39  (SEQ ID NO: 56) 59 ACPLVNPLCLMPGWGCA 114.5 ± 10.78 (SEQ ID NO: 57) 60 ACPLVNPLCMIGNWTCA 96.2 ± 0.59 (SEQ ID NO: 58) 61 ACPLVNPLCLMTGWSCA 241.5 ± 44.1  (SEQ ID NO: 59) 62 ACPLVNPLCMMGGWKCA  67.1 ± 19.21 (SEQ ID NO: 60) 63 ACPLVNPLCLYGSWKCA 59.05 ± 28.32 (SEQ ID NO: 61) 64 ACPLVNPLCLHPGWTCA 30 n = 1 (SEQ ID NO: 62) 65 ARDCPLVNPLCLHPGWTCA 6.05 ± 1.38 39.1 ± 0.39 (SEQ ID NO: 63) 66 (β-Ala)-Sar₁₀-A(HArg) 4.94 ± 1.41  57.6 ± 24.86 (BCY6099) DC(HyP)LVNPLCLHP (D-Asp)W(HArg)C (SEQ ID NO: 2) 67 (β-Ala)-Sar₁₀-A(HArg) 8.51 ± 0.17  61.7 ± 15.48 (BCY6014) DCPLVNPLCLHPGWTC ((β-Ala)-Sar₁₀- (SEQ ID NO: 11) 68 Ac-ARDCPLVNPLCLHPGWTCA- 19.3 ± 4.92 166.5 ± 30.38 Sar₆-(D-K) (Ac-(SEQ ID NO: 63)- Sar₆-(D-K)) 69 Ac-A(HArg) 17.5 ± 0.98 164.5 ± 2.94  DCPLVNPLCLHPGWTCA- Sar₆-(D-K) (Ac-(SEQ ID NO: 11)- A-Sar₆-(D-K)) 70 RPACPLVNPLCLHPGWTCA 10.06 ± 2.96  (SEQ ID NO: 64) 71 RPPCPLVNPLCLHPGWTCA 11.11 ± 2.25  (SEQ ID NO: 65) 72 KHSCPLVNPLCLHPGWTCA 11.92 ± 6.04  (SEQ ID NO: 66) 73 ACPLVNPLCLHPGWTCLHG 1.98 ± 0.49 72.7 ± 1.09 (SEQ ID NO: 67) 74 Ac-CPLVNPLCLHPGWTCLHG 1.76 ± 0.54 (Ac-(SEQ ID NO: 12)) 75 (β-Ala)-Sar₁₀- 2.48 ± 0.27  18 ± 1.18 ACPLVNPLCLHPGWTCLHG ((β-Ala)-Sar₁₀- (SEQ ID NO: 67)) 76 (β-Ala)-Sar₁₀- 10.01 ± 1.55  75.15 ± 14.41 ACPLVNPLCLHPGWTCL(D-His)G ((β-Ala)-Sar₁₀- (SEQ ID NO: 68)) 77 Ac-CPLVNPLCLHPGWTCLHG- 5.41 ± 0.86 48.23 ± 15.72 (BCY6019) Sar₆-(D-K) (Ac-(SEQ ID NO: 12)- Sar₆-(D-K)) 78 Ac-CPLVNPLCLHPGWTCL 15.6 ± 4.7  115.03 ± 41.16  (D-His)G-Sar₆-(D-K) (Ac-(SEQ ID NO: 13)- Sar₆-(D-K)) 79 ACPLVNPLCLHPG(2Nal)TCLHG   162 ± 17.64 (SEQ ID NO: 69) 80 RHDCPLVNPLCLLPGWTCA 7.11 ± 0.72 (SEQ ID NO: 70) 81 TPRCPLVNPLCLMPGWTCA  9.8 ± 2.61 (SEQ ID NO: 71) 82 ACPLVNPLCLAPGWTCA 46.2 n = 1 (SEQ ID NO: 72) 83 ACPLVNPLCLAPGWTCSRS 7.05 ± 1.11 (SEQ ID NO: 73) 84 ACPLVNPLCLEPGWTCA 53.9 n = 1 (SEQ ID NO: 74) 85 ACPLVNPLCLEPGWTCAKR 10.95 ± 1.6   (SEQ ID NO: 75) 86 ACPLVNPLCLHPGWSCA 56.15 ± 11.27 (SEQ ID NO: 76) 87 ACPLVNPLCLHPGWSCRGQ 2.57 ± 0.63 18.6 ± 0.59 (BCY6026) (SEQ ID NO: 77) 88 Ac-CPLVNPLCLHPGWSCRGQ 1.64 ± 0.75 (Ac-(SEQ ID NO: 14)) 89 β13-Ala)-Sar₁₀- 2.86 ± 1.29 29.55 ± 4.61  ACPLVNPLCLHPGWSCRGQ ((β-Ala)-Sar₁₀- (SEQ ID NO: 77) 90 (β-Ala)-Sar₁₀- 5.41 ± 0.67 47.05 ± 11.47 ACPLVNPLCLHPGWSC(HArg)GQ (β-Ala)-Sar₁₀- (SEQ ID NO: 78)) 91 Ac-CPLVNPLCLHPGWSCRGQ- 5.98 ± 1.42 49.87 ± 14.44 (BCY6042) Sar₆-(D-K) (Ac-(SEQ ID NO: 14)- Sar₆-(D-K)) 92 Ac-CPLVNPLCLHPGWSC(HArg) 10.56 ± 6.56  75.27 ± 21.72 GQ-Sar₆-(D-K) (Ac-(SEQ ID NO: 15)- Sar₆-(D-K)) 93 ACPLVNPLCLHPG(2Nal)SCRGQ    228 ± 103.88 (SEQ ID NO: 79) 94 ACPLVNPLCLTPGWTCTNT 13.25 ± 4.05  (SEQ ID NO: 80) 95 ACPMVNPLCLHPGWKCA 11.91 ± 3.73  (SEQ ID NO: 81) 96 ACPMVNPLCLTPGWICA 16.07 ± 4.58  (SEQ ID NO: 82) 97 ACPMVNPLCLHPGWTCA   20 ± 1.02 (SEQ ID NO: 83)

TABLE 5 Biological Assay Data for TATA Peptide Ligands of the Invention (Competition Binding Assay) Ki, nM ± 95% CI Human EphA2 Mouse EPhA2 Bicycle Fluorescent peptide Compound Reference Reference Number Sequence Compound C Compound C 98 (β-Ala)-Sar₁₀-H(D-Asp)VT- 251.5 ± 73.5 C(Aib)(1Nal)G(Aib)F(1Nal) CP(tBuGly)N(HArg)P(D-Asp)C ((β-Ala)-Sar₁₀-(SEQ ID NO: 84))

TABLE 6 Biological Assay Data for Peptide Ligands of the Invention (BDC competition binding data with TATA Scaffolds) Ki, nM Human EphA2 Mouse EphA2 BDC Fluorescent Peptide Compound Bicycle General Reference Reference Number precursor Formula Compound C Compound C BCY6027 BCY6099 Formula (A) 10.23 BCY6028 BCY6099 Formula (B) 13.04 BCY6031 BCY6014 Formula (A) 12.62 34.70 BCY6032 BCY6014 Formula (B) 11.42 35.90

TABLE 7 Selectivity Data for Peptide Ligands of the Invention (Selectivity Direct Binding Assay) Human Bicycle & rat & Compound mouse rat dog mouse rat human mouse rat Number EphA2 EphA2 EphA2 EphA3 EphA3 EphA4 EphA4 EphB1 2 516.5 ± 236.18  210 ± 1.96 >1000 >1000 >1000 10890 n = 1 7 216 252.5 ± 6.86  9 >3000 11 >3000 12 >3000 13 >3000 14 >3000 human Bicycle human Carbonic Compound Human Human Human Human Factor anhydrase human Number EphB4 EphA7 EphA6 EphA1 Xlla 9 CD38 2 >6000 7 9 11 12 13 14

Study 2: Fluorescence Polarisation Measurements (Alternative Protocol)

(a) Competition Binding

Peptides without a fluorescent tag were tested in competition with a peptide with a fluorescent tag and a known Kd (Table 9). Five μL of increasing (2 fold) concentrations of test compound was added to the plate followed by 10 μL of EphA2 protein (Table 8) at a fixed concentration which was dependent on the fluorescent peptide used (Table 9), then 10 μL fluorescent peptide added. Buffer was assay buffer as above with DMSO<1%. Measurements were conducted on a BMG PHERAstar FS equipped with an “FP 485 520 520” optic module which excites at 485 nm and detects parallel and perpendicular emission at 520 nm. The PHERAstar FS was set at 25° C. with 200 flashes per well and a positioning delay of 0.1 second, with each well measured at 5 to 10 minute intervals for 60 minutes. Alternatively, measurements were done on at similar time intervals on a Perkin Elmer Envision equipped with FITC FP Dual Mirror, FITC FP 480 excitation filter and FITC FP P-pol 535 and FITC FP S-pol emission filters with 30 flashes and a G-Factor of 1.2. Data analysis was in Systat Sigmaplot version 12.0 or 13.0 where the mP values at 60 minutes were fit to a user defined cubic equation to generate a Ki value:

f=ymin+(ymax−ymin)/Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}3){circumflex over ( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))/((3*Klig)+((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Kig*Kcomp)){circumflex over ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}3){circumflex over ( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))). “Lig”, “KLig” and “Prot” were all defined values relating to: fluorescent peptide concentration, the Kd of the fluorescent peptide and EphA2 concentration respectively.

TABLE 8 Eph receptors and source Receptor Catalogue (domain) Species Format/tag Supplier number EphA2 (Ecto) Human C-terminal R&D systems 3035-A2 polyHis EphA2 (Ecto) Human C- In-house N/A terminal polyHis EphA2 (Ecto) Mouse C- Sino 50586-M08H terminal polyHis Biological EphA2 (ligand Rat C- In-house N/A binding) terminal polyHis

TABLE 9 Final concentrations of fluorescent peptide and EphA2 as used with competition binding assays Concen- Concen- Concen- Concen- tration of tration tration tration Fluorescent fluorescent of human of mouse of rat EphA2 peptide peptide (nM) EphA2 (nM) EphA2 (nM) (nM) Reference 0.8 2.4 or 25 50 or 15 nM 25 Compound C

Certain peptide ligands and bicycle drug conjugates of the invention were tested in the above mentioned competition binding assay and the results are shown in Tables 10 to 11:

TABLE 10 Competition Binding with Selected Bicyclic Peptides Human Ki Mouse Ki Bicycle No. (nM) (nM) Rat Ki (nM) BCY6009 12.7 26.7 18.0 (Compound 108) BCY6014 14.5 39.6 24.4 (Compound 67) BCY6017 8.3 (Compound 109) BCY6018 13.1 (Compound 110) BCY6019 6.4 16.0 (Compound 77) BCY6026 4.4 (Compound 87) BCY6042 6.7 (Compound 91) BCY6059 43.2 (Compound 106) BCY6099 2.7 4.5 1.9 (Compound 66) BCY6101 9.7 6.9 (Compound 101) BCY6102 14.6 25.1 (Compound 102) BCY6103 14.8 20.8 (Compound 100) BCY6104 5.1 19.8 (Compound 99) BCY6137 2.2 (Compound 105) BCY6138 566.0 (Compound 104) BCY6139 5.7 (Compound 103) BCY6141 90.4 (Compound 112) BCY6152 23.3 (Compound 111) BCY6153 18.2 (Compound 113) BCY6160 14.0 (Compound 107) BCY6039 9.4 BCY6105 8.86 BCY6106 12.9 BCY6175 1 BCY6107 19.18

The results from the competition binding assay in Table 10 show that Bicycle peptides targeting human EphA2 (BCY6014 and BCY6099) bind with high affinity to mouse and rat EphA2. Similarly, BCY6019 binds to both human and mouse EphA2. These results show that certain peptides of the invention can be used in in vivo mouse and rat efficacy and toxicology models.

TABLE 11 Competition Binding with Selected Bicycle Drug Conjugates (BDCs) Mouse Bicycle Human Ki Rat Ki ID Ki (nM) (nM) (nM) BCY6061 12.0 32.3 14.2 BCY6174 1.7 3.9 3.0 BCY6029 2.3 BCY6033 9.9 34.2 13.4 BCY6037 7.3 BCY6049 8.8 28.1 BCY6053 48.2 29.7 BCY6122 13.7 10.4 BCY6136 1.9 5.5 3.2 BCY6030 5.6 BCY6034 5.9 35.9 BCY6038 2.8 BCY6050 168.1 62.2 BCY6054 53.6 73.6 BCY6027 10.2 BCY6031 12.5 35.1 20.0 BCY6035 15.2 BCY6047 53.2 34.2 BCY6051 54.0 43.6 BCY6134 7.4 12.6 BCY6135 2.4 5.0 2.9 BCY6154 8.0 BCY6155 12.5 BCY6063 7.8 66.8 BCY6028 13.0 BCY6032 11.4 35.9 BCY6036 18.6 BCY6048 120.7 87.2 BCY6052 30.5 27.1 BCY6064 12.5 40.7 BCY6162 44.9 BCY6082 10.5 34.1 13.9 BCY6150 17.9 BCY6151 9.0 BCY6161 2.1 BCY6173 1.7 4.3 2.5 BCY6077 6.5 25.3 BCY6055 15.8 BCY6062 12.9 20.3

Table 11 shows that certain Bicycle Drug Conjugates of the invention exhibit excellent cross reactivity between human, mouse and rodent EphA2. Peptides of the invention can therefore be used in mouse and rat efficacy and toxicology in vivo models.

(b) SPR Measurements

Non-Fc fusion proteins were biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin for 1 hour in 4 mM sodium acetate, 100 mM NaCl, pH 5.4 with a 3× molar excess of biotin over protein. The degree of labelling was determined using a Fluorescence Biotin Quantification Kit (Thermo) after dialysis of the reaction mixture into PBS. For analysis of peptide binding, a Biacore T200 instrument was used utilising a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25° C. with HBS—N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μl/min. For capture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5):HBS—N (1:1). Buffer was changed to PBS/0.05% Tween 20 and biotinylated EphA2 was captured to a level of 500-1500 RU using a dilution of protein to 0.2 μM in buffer. A dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5% with a top peptide concentration was 50 or 100 nM and 6 further 2-fold dilutions. The SPR analysis was run at 25° C. at a flow rate of 90 μl/min with 60 seconds association and 900-1200 seconds dissociation. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using simple 1:1 binding model allowing for mass transport effects where appropriate.

For binding of Bicycle Drug Conjugates a Biacore 3000 instrument was used. For biotinylated proteins immobilisation levels were 1500 RU and the top concentration was 100 nM. Otherwise the method was the same as described above using either the CMD500D or a CM5 chip (GE Healthcare). For the Fc-tagged proteins, a CM5 chip was activated as described above and then goat anti-human IgG antibody (Thermo-Fisher H10500) was diluted to 20 μg/ml in 10 mM sodium acetate pH5.0 and captured to approximately 3000 RU. The surface was then blocked as described above. Subsequent capture of the Fc-tagged proteins was carried out to obtain approximately 200-400 RU of the target protein. The proteins used are described below. All proteins were reconstituted as per manufacturer's suggested buffers and concentrations and captured using 5-10 μg/ml protein in PBS/0.05% Tween 20.

TABLE 12 Catalogue Receptor Species Format/tag Supplier number EphA1 Human Fc fusion Sino 15789-H02H Biologics EphA2 Human 0.95 mol In house N/A biotin/monomer EphA2 Mouse Fc fusion R&D 639-A2 Systems EphA2 Rat 1.4 mol biotin/ In house N/A monomer EphA3 Human Fc fusion R&D 6444-A3 Systems EphA3 Mouse Fc fusion Sino 51122-M02H Biologics EphA3 Rat Fc fusion Sino 80465-R02H Biologics EphA4 Human Fc fusion Sino 11314-H03H Biologics EphA4 Mouse Fc fusion Sino 50575-M02H Biologics EphA4 Rat Fc fusion Sino 80123-R02H Biologics EphA5 Human 3.1 mol R&D 3036-A5 biotin/monomer Systems EphA6 Human Fc fusion R&D 5606-A6 Systems EphA7 Human Fc fusion R&D 6756-A7 Systems EphB1 Rat Fc fusion R&D 1596-B1 Systems EphB4 Human Fc fusion Sino 10235-H02H Biologics

Certain peptide ligands and bicycle drug conjugates of the invention were tested in the above mentioned competition binding assay and the results are shown in Tables 13 to 15:

TABLE 13 SPR Binding Analysis with Selected Bicyclic Peptides and Bicycle Drug Conjugates of the Invention Human Mouse Rat Bicycle/BDC K_(D) K_(off) t_(1/2) K_(on) K_(D) K_(off) t_(1/2) K_(on) K_(D) K_(off) t_(1/2) K_(on) No. (nM) (s-1) (min) (M-1s-1) (nM) (s-1) (min) (M-1s-1) (nM) (s-1) (min) (M-1s-1) BCY6026 1.02 1.02E−03 11.3 9.92E+05 BCY6031 1.99 4.95E−03 2.3 2.49E+06 BCY6032 2.10 5.27E−03 2.2 2.52E+06 BCY6033 3.41 3.43E−03 3.5 9.99E+05 21.8 6.37E−03 1.8 2.92E+05 166 4.42E−03 2.6 2.67E+04 BCY6034 1.64 3.65E−03 3.2 2.23E+06 BCY6082 2.42 2.42E−03 4.8 9.87E+05 18.3 5.97E−03 1.9 3.27E+05 28.8 3.64E−03 3.2 1.26E+05 BCY6136 1.17 1.15E−03 10.0 9.86E+05 2.53 1.11E−03 10.4 4.37E+05 2.96 9.11E−04 12.6 3.07E+05 BCY6173 0.73 1.24E−03 9.3 1.69E+06 2.95 1.14E−03 10.1 3.86E+05 1.10 9.60E−04 12.0 8.81E+05

Table 13 details binding affinities and kinetic parameters (Koff and Kon) for binding of selected Bicycle Drug Conjugates to human EphA2 determined using the SPR assay.

TABLE 14 SPR Binding Analysis with Selected Bicycle Drug Conjugates of the Invention with Human Eph Homologs BDC No. EphA1 EphA3 EphA4 EphA5 EphA6 EphA7 EphB4 BCY6033 no binding @ no binding @ no binding @ no binding @ no binding @ no binding @ no binding @ 5 μM 5 μM 5 μM 25 μM 20 μM 20 μM 20 μM BCY6082 no binding @ no binding @ no binding @ no binding @ no binding @ no binding @ no binding @ 5 μM 5 μM 5 μM 25 μM 20 μM 20 μM 20 μM BCY6136 no binding @ no binding @ no binding @ no binding @ no binding @ no binding @ no binding @ 5 μM 5 μM 5 μM 25 μM 20 μM 20 μM 20 μM BCY6173 no binding @ no binding @ no binding @ no binding @ no binding @ no binding @ no binding @ 5 μM 5 μM 5 μM 25 μM 20 μM 20 μM 20 μM

Table 14 illustrates binding results with four Bicycle Drug Conjugates (BCY6033, BCY6082, BCY6136 and BCY6173) in the SPR assay with closely related human Ephrin homologs. The results show that compounds of the invention exhibit no significant binding to closely related human homologs: EphA1, EphA3, EphA4, EphA5, EphA6, EphA7 and EphB4.

TABLE 15 SPR Binding Analysis with Selected Bicycle Drug Conjugates of the Invention with Mouse and Rat Eph Orthologs BDC No. Mouse EphA3 Mouse EphA4 Rat EphA3 Rat EphB1 BCY6033 no binding @ no binding @ no binding @ no binding @ 20 μM 20 μM 20 μM 20 μM BCY6082 no binding @ no binding @ no binding @ no binding @ 20 μM 20 μM 20 μM 20 μM BCY6136 no binding @ no binding @ no binding @ no binding @ 20 μM 20 μM 20 μM 20 μM BCY6173 no binding @ no binding @ no binding @ no binding @ 20 μM 20 μM 20 μM 20 μM

The results in Table 15 show that certain Bicycle Drug Conjugates of the invention (BCY6033, BCY6082, BCY6136 and BCY6173) are also selective for mouse and rat EphA2 and exhibit no significant binding to closely related homologs: mouse EphA3 and EphA4; and rat EphA3 and EphB1.

Studies 3 and 7-23

In each of Studies 3 and 7-23, the following methodology was adopted for each study:

(a) Materials

(i) Animals and Housing Condition

Animals

-   -   Species: Mus Musculus     -   Strain: Balb/c nude or CB17-SCID     -   Age: 6-8 weeks     -   Body weight: 18-22 g     -   Number of animals: 9-90 mice     -   Animal supplier: Shanghai Lingchang Biotechnology Experimental         Animal Co. Limited Housing condition     -   The mice were kept in individual ventilation cages at constant         temperature and humidity with 3-5 animals in each cage.         -   Temperature: 20-26° C.         -   Humidity 40-70%.     -   Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.         The bedding material is corn cob, which is changed twice per         week.     -   Diet: Animals had free access to irradiation sterilized dry         granule food during the entire study period.     -   Water: Animals had free access to sterile drinking water.     -   Cage identification: The identification labels for each cage         contained the following information: number of animals, sex,         strain, the date received, treatment, study number, group number         and the starting date of the treatment.     -   Animal identification: Animals were marked by ear coding.

(ii) Test and Positive Control Articles

Physical Molecular Storage Number Description Weight Purity Condition BCY6031 Lyophilised powder 3878.92 97.99% Stored at −80° C. BCY6033 Lyophilised powder 4260.01 99.12% Stored at −80° C. BCY6082 Lyophilised powder 3911.04  96.8% Stored at −80° C. BCY6135 Lyophilised powder 4021 95.14% Stored at −80° C. BCY6136 Lyophilised powder 4402.23 97.5- Stored at −80° C.  98.6% BCY6173 Lyophilised powder 4101.15 95.80% Stored at −80° C. BCY6174 Lyophilised powder 4537 99.50% Stored at −80° C. BCY6175 Lyophilised powder 4492.29 96.20% Stored at −80° C. BCY8245 Lyophilised powder 4173.85 99.30% Stored at −80° C. BCY8781 Lyophilised powder 4173.83 99.00% Stored at −80° C. ADC Solution (10.47 mg/ — >99.00%   Stored at −80° C. (MEDI- ml concentration) 547)¹ ¹Full details of MEDI-547 (a fully human monoclonal antibody 1C1 (recognizing both human and murine EphA2) conjugated to MMAF via an mc linker) are described in Jackson et al (2008) Cancer Res 68, 9367-74.

(b) Experimental Methods and Procedures

(i) Observations

All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec, following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). At the time of routine monitoring, the animals were daily checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption (by looking only), body weight gain/loss, eye/hair matting and any other abnormal effect as stated in the protocol. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset.

(ii) Tumor Measurements and the Endpoints

The major endpoint was to see if the tumor growth could be delayed or mice could be cured. Tumor volume was measured three times weekly in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V=0.5 a×b² where a and b are the long and short diameters of the tumor, respectively. The tumor size was then used for calculations of T/C value. The T/C value (in percent) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively, on a given day.

TGI was calculated for each group using the formula: TGI (%)=[1−(T_(i)−T₀)/(V_(i)−V₀)]×100; T_(i) is the average tumor volume of a treatment group on a given day, T₀ is the average tumor volume of the treatment group on the day of treatment start, V_(i) is the average tumor volume of the vehicle control group on the same day with T_(i), and V₀ is the average tumor volume of the vehicle group on the day of treatment start.

(iii) Sample Collection

At the end of study the tumors of all groups were collected for FFPE.

(iv) Statistical Analysis

Summary statistics, including mean and the standard error of the mean (SEM), are provided for the tumor volume of each group at each time point.

Statistical analysis of difference in tumor volume among the groups was conducted on the data obtained at the best therapeutic time point after the final dose.

A one-way ANOVA was performed to compare tumor volume among groups, and when a significant F-statistics (a ratio of treatment variance to the error variance) was obtained, comparisons between groups were carried out with Games-Howell test. All data were analyzed using GraphPad Prism 5.0. P<0.05 was considered to be statistically significant.

Study 3: In Vivo Efficacy in the LU-01-0046 PDX Model

Cancer cell lines (CCL) are originally derived from patient tumors, but acquire the ability to proliferate within in vitro cell cultures. As a result of in vitro manipulation, CCL that have been traditionally used in cancer research undergo genetic transformations that are not restored when cells are allowed to grow in vivo. Because of the cell culturing process cells that are better adapted to survive in culture are selected, tumor resident cells and proteins that interact with cancer cells are eliminated, and the culture becomes phenotypically homogeneous. Researchers are beginning to attribute the reason that only 5% of anti-cancer agents are approved by the Food and Drug Administration after pre-clinical testing to the lack of tumor heterogeneity and the absence of the human stromal microenvironment. Specifically, CCL-xenografts often are not predictive of the drug response in the primary tumors because CCL do not follow pathways of drug resistance or the effects of the microenvironment on drug response found in human primary tumors. To overcome these problems, the inventors have used PDX models to improve the predictive power of pre-clinical models.

PDX are created when cancerous tissue from a patient's primary tumor is implanted directly into an immunodeficient mouse. PDX can maintain patient histology, including the presence of non-tumor cells (eg stromal cells) and thus better mimic the tumor microenvironment. In general PDX are therefore more reflective of the heterogeneity and histology of primary tumors than CCL-xenografts.

BCY6031 was screened in a primary adenocarcinoma PDX xenograft (LU-01-0046) derived from a patient with non-small cell lung carcinomas (NSCLC). LU-01-0046 has been shown to express high levels of EphA2 using RNA sequencing. BCY6031 exhibited excellent efficacy in the LU-01-0046 model and is therefore a promising novel therapy for the treatment of non-small cell lung cancer.

(a) Treatment Arms

The experiment was designed to compare tumour growth in vehicle treated animals and animals treated with BCY6031 at 5 mg/kg qw for four weeks.

TABLE 16 Dose Dosing Gr n Treatment (mg/kg) Volume (μl/g) Dosing Route Schedule 1 6 Vehicle — 10 i.v. biw*1 week 2 3 BCY6031 5 10 i.v. qw*4 weeks Note: n: animal number; Dosing volume: adjust dosing volume based on body weight.

(b) Experimental Method

(i) PDX Information

TABLE 17 Model Cancer EPH2 Name Type Tumor growth speed Array RSQ expression LU-01- NSCLC Tumor size can reach 6.790 31.312 High 0046 1000 mm³ in 40 days after tumor inoculation

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously in the right flank with an approximately 30 mm³ LU-01-0046 tumor fragment. Drug treatment was started when the average tumor volume reached 943 mm³. The test article, route of administration, dosing frequency and the animal numbers in each group are described above.

(iii) Testing Article Formulation Preparation

TABLE 18 Test article Dose(mg/kg) Formulation Vehicle — 50 mM Acetate, 10% Sucrose pH5 (without DMSO) BCY6031 5 Dissolve 4.59 mg BCY6031 into 4.498 ml formulation buffer to get the 1 mg/ml BCY6031 stock solution; Dilute 450 ul 1 mg/ml BCY6031 with 450 μl formulation buffer.

(c) Results

(i) Mortality, Morbidity, and Body Weight Gain or Loss

Animal body weight was monitored regularly as an indirect measure of toxicity. Body weight change in female Balb/C nude mice bearing LU-01-0046 tumor dosed with BCY6031 is shown in FIG. 1 .

(ii) Tumor Growth Curve

The tumor growth curve is shown in FIG. 2 .

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6031 in the PDX model LU-01-0046 was calculated based on tumor volume measurements on day 7 after the start of treatment.

TABLE 19 Tumor growth inhibition analysis (T/C and TGI) on Day 7 Tumor Volume T/C^(b) TGI Group Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, biw 2191 ± 473 — — — 2 BCY6031,  463 ± 158 21.1 138.6 p < 0.05 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Discussion

The study evaluated the therapeutic efficacy of BCY6031 in the LU-01-0046 PDX model. The measured body weights are shown in the FIG. 1 . Tumor volumes of the treatment group at various time points are shown in Table 19 and FIG. 2 .

The mean tumor size of vehicle treated mice reached 2191 mm³ on day 7. BCY6031 at 5 mg/kg produced potent antitumor activity with tumor measured as 463 mm³ (TGI=138.6%, p<0.05) by day 7. Furthermore, the BCY6031 treatment completely eradicated the tumors from day 32 and no tumour regrowth occurred following dosing suspension on day 28. BCY6031 gave rise to no significant body weight loss (FIG. 1 ) and there were no adverse clinical observations on drug treated mice throughout the study.

Study 4: In Vivo Efficacy of BCY6136 in CDX Xenograft Models

The study evaluated the therapeutic efficacy of BCY6136 in three Cancer Cell Line Derived (CDX) models: the HT1080 fibrosarcoma line, the MDA-MB-231 triple negative breast cancer line and the NCI-H1975 non-small cell lung cancer (NSCLC) line.

(a) Experimental Method

Balb/c mice were inoculated subcutaneously with tumour cells at the right flank and drug treatment started when the average the average tumour volume reached between 150 and 200 mm³. Tumour measurements and statistical analysis were performed as described above. Tumour bearing animals were treated once weekly with BCY6136 or vehicle.

(b) Discussion

FIGS. 4-6 show that BCY6136 is effective in breast, lung and fibrosarcoma xenograft models following once weekly dosing.

The HT1080 Fibrosarcoma Model:

In the HT1080 model complete regression of tumour growth was achieved by day 14 following once weekly dosing with BCY6136 on days 0 and 7 at 3 and 5 mg/kg (FIG. 4 ). Once weekly dosing with BCY6136 at 2 mg/kg on days 0 and 7 gave rise to tumour stasis (partial regression) (FIG. 4 ). BCY6136 treatment gave rise to no significant body weight loss (FIG. 4 inset) and there were no adverse clinical observations on drug treated mice throughout the study.

The NCI-H1975 NSCLC Model:

Complete regression of tumour growth in the NCI-H1975 model was observed by around day 28 following 2 and 3 mg/kg once weekly dosing with BCY6136 (FIG. 5 ). Following dosing cessation on day 35 no tumour regrowth was observed in the 3 mg/kg treated animals from day 35 to day 72 when the 3 mg/kg arm measurements ended (FIG. 5 ). Dosing with BCY6136 at 2 mg/kg gave rise to complete regression in this model from around day 28. Following dosing cessation on day 35 there was no tumour regrowth until around day 51 at the 2 mg/kg dose. At this dose level moderate tumour re-growth was observed from around day 51 until study termination on day 77. 1 mg/kg treatment with BCY6136 gave rise to tumour stasis (partial regression) (FIG. 5 ). BCY6136 treatment gave rise to no significant body weight loss (FIG. 5 inset) and there were no adverse clinical observations on drug treated mice throughout the study.

The MDA-MB-231 Breast Model:

Tumour stasis (partial regression) was observed in the MDA-MB231 model following once weekly dosing at 2 and 3 mg/kg from days 0 to day 45 (FIG. 6 ). Some body weight loss (attributed to tumour burden) was observed in the 2 mg/kg treated animals (FIG. 6 inset).

These results demonstrate that BCY6136 gives rise to profound tumour growth inhibition in mice implanted with fibrosarcoma, breast and lung CDX xenografts following once daily dosing.

Study 5: Safety Studies in the Rat

Six (6) female rats were randomly assigned to 3 groups of 2 rats/group to determine the toxicity of BCY6136, following administered by IV bolus injection at 5, 7.5 and 10 mg/kg on days 1 and 8. The study was terminated on day 15.

No significant effects on coagulation parameters (Prothrombin time (sec), Activated partial thromboplastin time (sec) or Fibroginogen levels (g/L) were observed on days 2, 12 and 15 (data not shown). No in-life bleeding events were reported and no evidence of internal bleeding was detected following pathology examination.

Study 6: Safety Studies in the Cynomolgous Monkeys

Twenty eight day toxicology studies with BCY6136 we conducted in cynomolgous monkeys. BCY6136 was dosed at 1.0 and 2.0 mg/kg on days 1, 8, 15 and 22. Animals were euthanised and necropsied on day 29 (7 days after the final dose).

No significant effects on coagulation parameters relative to baseline were observed on days 18, 22 and 25 (data not shown) and day 29 (Table 20). No in-life bleeding events were reported and no evidence of internal bleeding was detected following pathology examination.

TABLE 20 Day 29 coagulation parameters following 1.0 and 2.0 mg/kg BCY6136 dosing to cynomolgus monkeys 1.0 mg/kg × 4 2.0 mg/kg × 4 Baseline Day 29 Baseline Day 29 PT(s) 13.4 11.7 9.4 9.7 PT(s) 11 9.2 11.2 11.0 APTT(s) 18.9 19.4 19.4 20.9 APTT(s) 16.1 15.7 18.7 18.2 FIB(g/L) 2.08 2.42 1.86 6.1 FIB(g/L) 2.28 2.35 1.82 3.1

Study 7: In Vivo Efficacy Study of BCY6033 and BCY6136 and ADC in Treatment of PC-3 Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6033 and BCY6136 in treatment of PC-3 xenograft.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule 1 Vehicle 3 — 10 iv qw 2 BCY6136 3 1 10 iv qw 3 BCY6136 3 2 10 iv qw 4 BCY6136 3 3 10 iv qw 5 ADC 3 3 10 iv qw 6 BCY6033 3 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The PC-3 tumor cells will be maintained in F12K medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells will be routinely subcultured twice weekly. The cells growing in an exponential growth phase will be harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse will be inoculated subcutaneously at the right flank with PC-3 (10*10⁶) tumor cells for tumor development. The animals will be randomized and treatment will be started when the average tumor volume reaches approximately 150 mm³. The test article administration and the animal numbers in each group are shown in the following experimental design table.

(iii) Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 50 mM Acetate/acetic acid pH 5 10% sucrose BCY6136 0.1 Dilute 90 μl 1 mg/ml BCY6136 stock with 810 μl vehicle buffer 0.2 Dilute 180 μl 1 mg/ml BCY6136 stock with 720 μl vehicle buffer 0.3 Dilute 270 μl 1 mg/ml BCY6136 stock with 630 μl vehicle buffer ADC 0.3 Dilute 26 μl 10.47 mg/ml ADC stock with 874 μl ADC buffer BCY6033 0.3 Dilute 270 μl 1 mg/ml BCY6033 stock with 630 μl vehicle buffer

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIGS. 7 to 9 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing PC-3 xenograft is shown in Table 21.

TABLE 21 Tumor volume trace over time Gr Treatment 0 2 4 7 9 11 14 16 18 21 1 Vehicle, qw 149 ± 9   235 ± 9   377 ± 9   718 ± 30  1126 ± 41  1431 ± 79   1792 ± 69   2070 ± 152  2 BCY6136, 150 ± 11  185 ± 25  228 ± 31  201 ± 17  183 ± 23  153 ± 38  137 ± 33  107 ± 32  64 ± 28 45 ± 23 1mpk, qw 3 BCY6136, 149 ± 18  179 ± 28  158 ± 22  137 ± 16  122 ± 15  114 ± 20  101 ± 16  79 ± 20 57 ± 19 42 ± 17 2 mpk, qw 4 BCY6136 149 ± 2   155 ± 8   144 ± 16  132 ± 20  107 ± 28  94 ± 23 83 ± 22 70 ± 27 38 ± 16 35 ± 17 3 mpk, qw 5 ADC 151 ± 27  203 ± 10  210 ± 12  189 ± 11  185 ± 16  190 ± 37  158 ± 36  124 ± 35  103 ± 27  74 ± 14 3 mpk, qw 6 BCY6033, 151 ± 33  214 ± 53  204 ± 51  192 ± 53  163 ± 43  151 ± 40  141 ± 39  116 ± 36  83 ± 28 63 ± 32 3mpk, qw Gr Treatment 23 25 28 30 32 35 37 39 42 1 Vehicle, qw 2 BCY6136, 35 ± 18 28 ± 14 37 ± 19 34 ± 17 42 ± 21 42 ± 23 43 ± 21 28 ± 14 18 ± 9 1mpk, qw 3 BCY6136, 21 ± 11 22 ± 12 22 ± 12 24 ± 12 33 ± 16 22 ± 11 26 ± 14 22 ± 12 16 ± 9 2 mpk, qw 4 BCY6136 21 ± 10 23 ± 12 27 ± 14 22 ± 11 24 ± 12 20 ± 11 27 ± 14 12 ± 6 12 ± 6 3 mpk, qw 5 ADC 53 ± 16 50 ± 22 46 ± 23 70 ± 35 78 ± 39 53 ± 27 60 ± 30 53 ± 27 40 ± 22 3 mpk, qw 6 BCY6033, 59 ± 31 44 ± 27 39 ± 24 40 ± 29 47 ± 32 41 ± 27 41 ± 30 34 ± 24 33 ± 27 3mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the PC-3 xenograft model was calculated based on tumor volume measurements at day 16 after the start of treatment.

TABLE 22 Tumor growth inhibition analysis Tumor P value Volume T/C^(b) TGI compare Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw 2070 ± 152 — — — 2 BCY6136,  107 ± 32 5.2 102.2 p < 0.001 1 mpk, qw 3 BCY6136,  79 ± 20 3.8 103.6 p < 0.001 2 mpk, qw 4 BCY6136,  70 ± 27 3.4 104.1 p < 0.001 3 mpk, qw 5 ADC,  124 ± 35 6.0 101.4 p < 0.001 3 mpk, qw 6 BCY6033,  116 ± 36 5.6 101.8 p < 0.001 3 mpk, qw ^(a)Mean ± SEM. bTumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the PC-3 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIGS. 7 to 9 and Tables 21 and 22.

The mean tumor size of vehicle treated mice reached 2070 mm³ on day 16. BCY6136 at 1 mg/kg, qw (TV=107 mm³, TGI=102.2%, p<0.001), BCY6136 at 2 mg/kg, qw (TV=79 mm³, TGI=103.6%, p<0.001) and BCY6136 at 3 mg/kg, qw (TV=70 mm³, TGI=104.1%, p<0.001) showed potent anti-tumor effect.

BCY6033 at 3 mg/kg, qw (TV=116 mm³, TGI=101.8%, p<0.001) and ADC at 3 mg/kg, qw (TV=124 mm³, TGI=101.4%, p<0.001) showed comparable anti-tumor effect.

In this study, animal body weight was monitored regularly. All mice maintained their body weight well.

Study 8. In Vivo Efficacy Study of BCY6136 in Treatment of PC-3 Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of PC-3 xenograft in Balb/c nude mice.

(b) Experimental Design

Dose Dosing Group Treatment (mg/kg) N^(a) Route Schedule  1 Vehicle — 4 i.v.  qw × 4 weeks  2 BCY6136 0.167 4 i.v.  qw × 4 weeks  3^(b) BCY6136 0.5 4 i.v.  qw × 4 weeks  4 BCY6136 1.5 4 i.v.  qw × 4 weeks  5^(b) BCY6136 0.5 4 i.v. q2w × 2 weeks  6^(b) BCY6136 1.5 4 i.v. q2w × 2 weeks  7 EphA2-ADC 0.33 4 i.v.  qw × 4 weeks  8 EphA2-ADC 1 4 i.v.  qw × 4 weeks  9 EphA2-ADC 3 4 i.v.  qw × 4 weeks 10^(c) Docetaxel 15 4 i.v.  qw × 4 weeks ^(a)N, the number of animals in each group. ^(b)After 4 weeks' treatment demonstrated in the experimental design table, the mice of group 3, 5 and 6 were treated with BCY6136 1.5 mg/kg qw from day 52 during the monitoring schedule. ^(c)Due to the severe body weight loss of the Docetaxel treated mice after the first dosing, the treatment was suspended for 2 weeks, then a lower dosage (Docetaxel, 10 mg/kg) was performed on day 28. After that, the mice were treated with BCY6136 1.5 mg/kg qw from day 42 to day 70.

(c) Experimental Methods and Procedures

(i) Cell Culture

The tumor cells were maintained in F-12K medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with PC-3 tumor cells (10×10⁶) in 0.2 ml of PBS for tumor development. 52 animals were randomized when the average tumor volume reached 454 mm³. The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Test Conc. article Purity (mg/ml) Formulation Vehicle — — 25 mM Histidine pH 7 10% sucrose BCY6136 98.6% — 50 mM Acetate 10% sucrose pH 5 1 Dissolve 2.70 mg BCY6136 in 2.662 ml Acetate buffer 0.3 Dilute 300 μl 1 mg/ml BCY6136 stock with 700 μl Acetate buffer¹ 0.15 Dilute 600 μl 0.3 mg/ml BCY6136 stock with 600 μl Acetate buffer 0.05 Dilute 200 μl 0.3 mg/ml BCY6136 stock with 1000 μl Acetate buffer 0.0167 Dilute 66.7 μl 0.3 mg/ml BCY6136 stock with 1133.3 μl Acetate buffer EphA2- — — 25 mM Histidine pH 5.5 ADC 0.033 Dilute 9.3 μl 4.24 mg/ml EphA2- ADC stock with 1191 μl His buffer 0.1 Dilute 28 μl 4.24 mg/ml EphA2- ADC stock with 1172 μl His buffer 0.3 Dilute 84.9 μl 4.24 mg/ml EphA2- ADC stock with 1115 μl His buffer Docetaxel — 10 Mix 0.5 ml 20 mg Docetaxel with 1.5 ml buffer 1.5 Dilute 180 μl 10 mg/ml Docetaxel stock with 1020 μl saline buffer ¹50 mM Acetate 10% sucrose pH 5 3.25 mM Histidine pH 5.5

(c) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve is shown in FIG. 10 .

(ii) Tumor Volume Trace

Mean tumor volume over time in male Balb/c nude mice bearing PC-3 xenograft is shown in Table 23.

TABLE 23 Tumor volume trace over time (Day 0 to day 20) Days after the start of treatment Gr. Treatment 0 2 4 6 8 10 13 15 17 20 1 Vehicle, qw 456 ± 25 648 ± 50 880 ± 23 1022 ± 29 1178 ± 118 1327 ± 133 1631 ± 93 1868 ± 90 2052 ± 139 2364 ± 102 2 BCY6136 450 ± 33 631 ± 55 695 ± 78  739 ± 39  850 ± 68  904 ± 73  975 ± 47 1089 ± 74 1124 ± 92 1188 ± 111 0.167 mpk, qw 3 BCY6136 451 ± 47 622 ± 96 519 ± 70  460 ± 55  398 ± 50  329 ± 38  260 ± 33  249 ± 33  231 ± 38  234 ± 42 0.5 mpk, qw 4 BCY6136 458 ± 49 587 ± 63 494 ± 54  363 ± 32  283 ± 32  237 ± 24  192 ± 13  164 ± 16  155 ± 20  131 ± 19 1.5 mpk, qw 5 BCY6136 454 ± 37 643 ± 25 531 ± 37  458 ± 33  411 ± 32  382 ± 49  430 ± 88  522 ± 124  560 ± 129  530 ± 147 0.5 mpk, q2w 6 BCY6136 452 ± 42 590 ± 75 457 ± 49  375 ± 44  328 ± 47  242 ± 63  206 ± 61  197 ± 62  182 ± 55  128 ± 36 1.5 mpk, q2w 1.5 mpk, qw 7 EphA2-ADC 457 ± 43 636 ± 57 712 ± 70  792 ± 78  870 ± 87  900 ± 58 1049 ± 66 1242 ± 123 1443 ± 12  1637 ± 181 0.33 mpk, qw 8 EphA2-ADC 450 ± 49 617 ± 48 673 ± 50  721 ± 61  782 ± 78  755 ± 67  840 ± 93  913 ± 91  978 ± 100  981 ± 100 1 mpk, qw 9 EphA2-ADC 452 ± 60 593 ± 98 643 ± 141  593 ± 106  433 ± 103  290 ± 81  268 ± 64  232 ± 60  225 ± 66  184 ± 62 3 mpk, qw 10 Docetaxel 453 ± 62 584 ± 72 632 ± 56  636 ± 48  568 ± 50  408 ± 31  374 ± 26  388 ± 36  361 ± 25  419 ± 31 15 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the PC-3 xenograft model was calculated based on tumor volume measurements at day 20 after the start of the treatment.

TABLE 24 Tumor growth inhibition analysis Tumor P value Volume T/C^(b) TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle  1 Vehicle, qw 2364 ± 102 — — —  2 BCY6136, 1188 ± 111 50.2 61.4 p < 0.001 0.167 mpk, qw  3 BCY6136,  234 ± 42 9.9 111.4 p < 0.001 0.5 mpk, qw  4 BCY6136,  131 ± 19 5.5 117.2 p < 0.001 1.5 mpk, qw  5 BCY6136,  530 ± 147 22.4 96.0 p < 0.001 0.5 mpk, q2w  6 BCY6136,  128 ± 36 5.4 117.0 p < 0.001 1.5 mpk, q2w  7 EphA2-ADC, 1637 ± 181 69.2 38.1 p < 0.001 0.33 mpk, qw  8 EphA2-ADC,  981 ± 100 41.5 72.2 p < 0.001 1 mpk, qw  9 EphA2-ADC,  184 ± 62 7.8 114.0 p < 0.001 3 mpk, qw 10 Docetaxel,  419 ± 31 17.7 101.8 p < 0.001 15 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(d) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the PC-3 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIG. 10 and Tables 23 and 24.

The mean tumor size of vehicle treated mice reached 2364 mm³ on day 20. BCY6136 at 0.167 mg/kg, qw (TV=1188 mm³, TGI=61.4%, p<0.001), 0.5 mg/kg, q2w (TV=530 mm³, TGI=96.0%, p<0.001), 0.5 mg/kg, qw (TV=234 mm³, TGI=111.4%, p<0.001) and 1.5 mg/kg, qw (TV=131 mm³, TGI=117.2%, p<0.001) produced significant anti-tumor activity in dose or dose-frequency dependent manner on day 20. BCY6136 at 1.5 mg/kg, q2w (TV=128 mm³, TGI=117.0%, p<0.001) produced comparable anti-tumor activity with BCY6136 1.5 mg/kg qw. Among them, the mice treated with BCY6136, 0.5 mg/kg qw or BCY6136, 0.5 mg/kg q2w showed obvious tumor relapse after ceasing the treatment, further treatment with BCY6136, 1.5 mg/kg qw from day 52 worked well on the tumor regression. The mice treated with BCY6136, 1.5 mg/kg q2w also showed tumor relapse after ceasing the treatment, but further dosing didn't work on complete tumor regression. The mice treated with BCY6136, 1.5 mpk qw didn't show any tumor relapse until day 48.

EphA2-ADC at 0.33 mg/kg, qw (TV=1637 mm³, TGI=38.1%, p<0.001), 1 mg/kg, qw (TV=981 mm³, TGI=72.2%, p<0.001) and 3 mg/kg, qw (TV=184 mm³, TGI=114.0%, p<0.001) produced significant anti-tumor activity in dose dependent manner on day 20. The mice treated with EphA2-ADC, 3 mg/kg qw didn't show any tumor relapse until day 59.

Docetaxel at 15 mg/kg, qw (TV=419 mm³, TGI=101.8%, p<0.001) produced significant anti-tumor activity but caused severe animal body weight loss. After ceasing the treatment, the mice showed obvious tumor relapse. The treatment with BCY6136, 1.5 mg/kg qw from day 42 worked well on tumor regression of these mice.

Study 9. In Vivo Efficacy Test of BCY6033, BCY6136 and BCY6082 in Treatment of NCI-H1975 Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCY6033, BCY6136 and BCY6082 in treatment of NCI-H1975 xenograft model in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule 1 Vehicle 3 — 10 iv qw 2 BCY6033 3 1 10 iv qw 3 BCY6033 3 2 10 iv qw 4 BCY6033 3 3 10 iv qw 5 BCY6136 3 1 10 iv qw 6 BCY6136 3 2 10 iv qw 7 BCY6136 3 3 10 iv qw 8 BCY6082 3 2 10 iv qw 9 BCY6082 3 5 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with NCI-H1975 tumor cells (10×10{circumflex over ( )}6) in 0.2 ml of PBS for tumor development. 36 animals were randomized when the average tumor volume reached 149 mm³. The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Dose Treatment (mg/ml) Formulation Vehicle 50 mM Acetate, 10% sucrose pH = 5 BCY6033 1 Dissolve 6.71 mg BCY6033 in 6.710 ml formulation buffer 0.3 Dilute 270 μl 1 mg/ml BCY6033 with 630 μl formulation buffer 0.2 Dilute 180 μl 1 mg/ml BCY6033 with 720 μl formulation buffer 0.1 Dilute 90 μl 1 mg/ml BCY6033 with 810 μl formulation buffer BCY6136 1 Dissolve 3.79 mg BCY6136 in 3.695 ml formulation buffer 0.3 Dilute 270 μl 1 mg/ml BCY6136 with 630 μl formulation buffer 0.2 Dilute 180 μl 1 mg/ml BCY6136 with 720 μl formulation buffer 0.1 Dilute 90 μl 1 mg/ml BCY6136 with 810 μl formulation buffer BCY6082 1 Weigh and dissolve 4.30 mg BCY6082 in 4.162 ml formulation buffer 0.5 Dilute 450 μl 1 mg/ml BCY6082 with 450 μl formulation buffer 0.2 Dilute 180 μl 1 mg/ml BCY6082 with 720 μl formulation buffer

(iv) Sample Collection

On PG-D23, we fixed the tumors of Group 1 for FFPE.

On PG-D44, we fixed the tumors of Group 2 and 5 for FFPE.

At the end of study, we the tumors of Group 6 for FFPE.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth are shown in FIGS. 11 to 13 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing NCI-H1975 xenograft is shown in Table 25 to 29.

TABLE 25 Tumor volume trace (PG-D0-PG-17) Days after the start of treatment Gr. Treatment 0 2 4 7 9 11 14 17 1 Vehicle, qw 148 ± 4   195 ± 11  297 ± 33  466 ± 64  732 ± 107 1028 ± 192  1278 ± 252  1543 ± 298  2 BCY6033, 149 ± 10  160 ± 4   207 ± 13  259 ± 49  330 ± 69  365 ± 83  341 ± 59  336 ± 54  1 mpk, qw 3 BCY6033, 149 ± 10  183 ± 11  276 ± 24  365 ± 42  405 ± 20  364 ± 19  319 ± 32  304 ± 33  2 mpk, qw 4 BCY6033, 149 ± 6   161 ± 4   207 ± 26  260 ± 21  270 ± 42  243 ± 52  187 ± 53  131 ± 43  3 mpk, qw 5 BCY6136, 150 ± 6   178 ± 20  232 ± 49  336 ± 43  400 ± 24  407 ± 42  299 ± 113 261 ± 127 1 mpk, qw 6 BCY6136, 150 ± 14  181 ± 26  237 ± 27  277 ± 36  297 ± 37  306 ± 55  256 ± 53  218 ± 49  2 mpk, qw 7 BCY6136, 148 ± 9   168 ± 10  231 ± 6   365 ± 16  390 ± 13  423 ± 42  319 ± 26  228 ± 16  3 mpk, qw 8 BCY6082, 148 ± 5   157 ± 4   223 ± 19  370 ± 84  447 ± 102 658 ± 188 906 ± 332 1123 ± 410  2 mpk, qw 9 BCY6082, 148 ± 6   176 ± 12  235 ± 19  378 ± 59  436 ± 68  510 ± 82  484 ± 78  491 ± 103 5 mpk, qw

TABLE 26 Tumor volume trace (PG-018-PG-035) Days after the start of treatment Gr. Treatment 18 21 23 25 28 30 33 35 1 Vehicle, qw 1864 ± 395  2371 ± 470  — — — — — — 2 BCY6033, 278 ± 71  306 ± 81  343 ± 86  366 ± 89  466 ± 115 481 ± 112 619 ± 170 780 ± 236 1 mpk, qw 3 BCY6033, 172 ± 25  95 ± 12 61 ± 6  39 ± 4  13 ± 1  12 ± 1  6 ± 3 6 ± 3 2 mpk, qw 4 BCY6033, 75 ± 15 29 ± 4  20 ± 6  13 ± 2  6 ± 0 4 ± 0 1 ± 0 2 ± 1 3 mpk, qw 5 BCY6136, 215 ± 113 205 ± 117 197 ± 113 200 ± 105 202 ± 112 202 ± 117 230 ± 142 241 ± 127 1 mpk, qw 6 BCY6136, 149 ± 31   99 ± 30 69 ± 22 42 ± 13 30 ± 10 16 ± 8  20 ± 9  4 ± 2 2 mpk, qw 7 BCY6136, 149 ± 17  94 ± 30 50 ± 15 41 ± 21 21 ± 8  6 ± 6 10 ± 6  3 ± 1 3 mpk, qw 8 BCY6082, 1199 ± 408  1528 ± 604  1978 ± 792  2499 ± 931  — — — — 2 mpk, qw 9 BCY6082, 471 ± 143 390 ± 133 368 ± 122 295 ± 102 227 ± 86  — — — 5 mpk, qw

TABLE 27 Tumor volume trace (PG-037-PG-053) Days after the start of treatment Gr. Treatment 37 39 42 44 46 49 51 53 2 BCY6033, 877 ± 188 945 ± 145 1258 ± 173  — — — — — 1 mpk, qw 3 BCY6033, 3 ± 1 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 2 mpk, qw 4 BCY6033, 0 ± 0 0 ± 0 0 ± 0 0 ± 0 1 ± 0 0 ± 0 1 ± 0 1 ± 0 3 mpk, qw 5 BCY6136, 277 ± 149 294 ± 159 351 ± 188 — — — — — 1 mpk, qw 6 BCY6136, 7 ± 4 2 ± 1 1 ± 0 3 ± 1 2 ± 1 3 ± 2 6 ± 3 14 ± 10 2 mpk, qw 7 BCY6136, 3 ± 3 2 ± 1 1 ± 0 0 ± 0 0 ± 0 0 ± 0 1 ± 0 1 ± 0 3 mpk, qw

TABLE 28 Tumor volume trace (PG-056~PG-074) Days after the start of treatment Gr. Treatment 56 58 60 63 65 67 70 72 74 3 BCY6033, 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 2 ± 1 4 ± 3 7 ± 6 — 2 mpk, qw 4 BCY6033, 1 ± 0 1 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 — 3 mpk, qw 6 BCY6136, 16 ± 11 27 ± 18 34 ± 23 45 ± 31 63 ± 40 71 ± 47 95 ± 70 111 ± 73  122 ± 75 2 mpk, qw 7 BCY6136, 1 ± 0 1 ± 0 1 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 — 3 mpk, qw

TABLE 29 Tumor volume trace (PG-D77~PG-D98) Days after the start of treatment Gr. Treatment 77 81 84 88 91 95 98 6 BCY6136, 208 ± 112 337 ± 123 501 ± 172 626 ± 182 856 ± 245 1035 ± 169 1266 ± 39 2 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6033, BCY6136 and BCY6082 in the NCI-H1975 xenograft model was calculated based on tumor volume measurements at day 21 after the start of treatment.

TABLE 30 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Gr Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 2371 ± 470 — — — 2 BCY6033,  306 ± 81 12.9  92.9 p < 0.001 1 mpk, qw 3 BCY6033,  95 ± 12  4.0 102.5 p < 0.001 2 mpk, qw 4 BCY6033,  29 ± 4  1.2 105.4 p < 0.001 3 mpk, qw 5 BCY6136,  205 ± 117  8.6  97.5 p < 0.001 1 mpk, qw 6 BCY6136,  99 ± 30  4.2 102.3 p < 0.001 2 mpk, qw 7 BCY6136,  94 ± 30  4.0 102.4 p < 0.001 3 mpk, qw 8 BCY6082, 1528 ± 604 64.4  37.9 p > 0.05 2 mpk, qw 9 BCY6082,  390 ± 133 16.4  89.1 p < 0.001 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6033, BCY6136 and BCY6082 in the NCI-H1975 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIGS. 11 to 13 and Tables 25 to 30.

The mean tumor size of vehicle treated mice reached 2371 mm³ on day 21. BCY6033 at 1 mg/kg (TV=306 mm³, TGI=92.9%, p<0.001), 2 mg/kg (TV=95 mm³, TGI=102.5%, p<0.001) and 3 mg/kg (TV=29 mm³, TGI=105.4%, p<0.001) produced dose-dependent antitumor activity. BCY6033 at 2 mg/kg and 3 mg/kg eradicated the tumors or regressed the tumor to small size, the treatments was suspended from day 35, and the tumors didn't show obvious re-growth in following 5-6 weeks monitoring.

BCY6136 at 1 mg/kg (TV=205 mm³, TGI=97.5%, p<0.001), 2 mg/kg (TV=99 mm³, TGI=102.3%, p<0.001) and 3 mg/kg (TV=94 mm³, TGI=102.4%, p<0.001) produced potent antitumor activity. BCY6136 at 2 mg/kg and 3 mg/kg eradicated the tumors or regressed the tumor to small size. The treatments was suspended from day 35, and the tumors in 3 mg/kg group didn't show obvious re-growth in following 5-6 weeks monitoring, however tumors in 2 mg/kg group showed obvious regrowth and didn't show significant tumor inhibition when resuming the dosing.

BCY6082 at 2 mg/kg (TV=1528 mm3, TGI=37.9%, p>0.05) didn't show obvious antitumor activity, BCY6082 at 5 mg/kg (TV=390 mm3, TGI=89.1%, p<0.001) produced significant antitumor activity.

In this study, one mouse treated with BCY6033 3 mg/kg lost over 15% bodyweight during the monitoring, other mice maintained the bodyweight well.

Study 10. In Vivo Efficacy Study of BCY6136 in the LU-01-0251 PDX Model in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in the LU-01-0251 PDX model in Balb/c nude mice.

(b) Experimental Design

Dose Dosing Dosing Group Treatment n (mg/kg) Volume (μl/g) Route Schedule 1 Vehicle 5 — 10 iv qw 2 BCY6136 5 1 10 iv qw 3 BCY6136 5 2 10 iv qw 4 BCY6136 5 3 10 iv qw 5 ADC 5 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0251 of tumor fragment (˜30 mm³) for tumor development. The treatment was started when the average tumor volume reached 174 mm³ for efficacy study. The test article administration and the animal number in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10% sucrose pH 5 BCY6136 0.3 Dissolve 6.11 mg BCY6136 in 20 ml Acetate buffer¹ 0.2 Dilute 940 μl 0.3 mg/ml BCY6136 stock with 470 μl Acetate buffer 0.1 Dilute 470 μl 0.3 mg/ml BCY6136 stock with 940 μl Acetate buffer ADC 0.3 Dilute 43 μl 10.47 mg/ml ADC stock with 1457 μl ADC buffer² ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5 ²ADC buffer: 20 mM Histidine pH 5.5

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 14 .

(ii) Tumor Volume Trace

Mean tumor volume on day 28 after the start of treatment in female Balb/c nude mice bearing LU-01-0251 xenograft is shown in Table 31.

TABLE 31 Tumor volume trace over time Group 2 Group 3 Group 4 Group 5 Group 1 BCY6136, BCY6136, BCY6136, ADC, Day Vehicle 1 mpk, qw 2 mpk, qw 3 mpk, qw 3 mpk, qw 0  174 ± 17  175 ± 15  174 ± 17 175 ± 14 174 ± 16 3  264 ± 33  230 ± 29  205 ± 21 187 ± 19 227 ± 12 7  403 ± 68  281 ± 55  154 ± 21 118 ± 13 239 ± 42 10  562 ± 83  370 ± 104 111 ± 19  72 ± 12 241 ± 46 14  777 ± 163 362 ± 104  62 ± 17  30 ± 5  191 ± 47 17 1021 ± 246 437 ± 136  46 ± 13  17 ± 3  139 ± 39 21 1472 ± 342 526 ± 167  30 ± 18   4 ± 3  101 ± 31 24 1790 ± 417 491 ± 132  32 ± 24   1 ± 1   70 ± 23 28 2208 ± 512 499 ± 128  32 ± 30   0 ± 0   39 ± 14

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 and ADC in the LU-01-0251 PDX model was calculated based on tumor volume measurements at day 28 after the start of the treatment.

TABLE 32 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Group Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 2208 ± 512 — — — 2 BCY6136,  499 ± 128 22.6  84.0 p < 0.001 1 mpk, qw 3 BCY6136,  32 ± 30  1.4 107.0 p < 0.001 2 mpk, qw 4 BCY6136,   0 ± 0  0.0 108.6 p < 0.001 3 mpk, qw 5 ADC,  39 ± 14  1.8 106.6 p < 0.001 3 mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 and ADC in LU-01-0251 PDX model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in the FIG. 14 and Tables 31 and 32.

In this study, the mean tumor volume of vehicle treated mice reached 2208 mm³ on day 28 after the start of treatment. BCY6136 at 1 mg/kg, qw (TV=499 mm³, TGI=84.0%, p<0.001), 2 mg/kg, qw (TV=32 mm³, TGI=107.0%, p<0.001) and 3 mg/kg, qw (TV=0 mm³, TGI=108.6%, p<0.001) produced dose-dependent anti-tumor activity. ADC at 3 mg/kg, qw (TV=39 mm³, TGI=106.6%, p<0.001) showed significant anti-tumor activity.

Study 11: In Vivo Efficacy Study of BCY6136 in the LU-01-0251 PDX Model in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in the LU-01-0251 POX model in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule 1 Vehicle 5 — 10 iv Qw*21 2 BCY6136 5 1 10 iv Qw*28  3^(a) BCY6136 5 2 10 iv Qw*70  4^(b) BCY6136 5 3 10 iv Qw*56  5^(c) ADC 5 3 10 iv Qw*70 a. The dosing schedule was kept from day 0 to day 70 for all the mice of this group, then the mouse 3-2 and mouse 3-4 were further dosed with BCY6136 3 mg/kg qw from day 77 while the treatment of the other 3 mice was suspended. The dosing schedule was kept from day 0 to day 56 for all the mice of this group. b. The dosing schedule was kept from day 0 to day 70 for all the mice of this group.

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0251 of tumor fragment (˜30 mm³) for tumor development. The treatment was started when the average tumor volume reached 960 mm³ for efficacy study. The test article administration and the animal number in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 25 mM Histidine 10% sucrose pH 7 BCY6136 0.3 0.3 mg/ml BCY6136 was prepared as in Study 10 hereinbefore 0.2 Dilute 940 μl 0.3 mg/ml BCY6136 stock with 470 μl His-buffer¹ 0.1 Dilute 470 μl 0.3 mg/ml BCY6136 stock with 940 μl His-buffer ADC 0.3 Dilute 43 μl 10.47 mg/ml ADC stock with 1457 μl ADC-buffer² ¹His-buffer: 25 mM Histidine 10% sucrose pH 7 ²ADC-buffer: 20 mM Histidine pH 5.5

(iii) Sample Collection

Tumor of mouse #3-2 was collected for FFPE on Day94. Tumors of mice #5-2 and 5-3 were collected and embed into 1 FFPE block on Day140.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 15 .

(ii) Tumor Volume Trace

Mean tumor volume on day 0 to day 28 after the start of treatment in female Balb/c nude mice bearing LU-01-0251 xenograft is shown in Table 33.

TABLE 33 Tumor volume trace over time Group 2 Group 3 Group 4 Group 5 Group 1 BCY6136, BCY6136, BCY6136, ADC, Day Vehicle 1 mpk, qw 2 mpk, qw 3 mpk, qw 3 mpk, qw  0  962 ± 102  963 ± 97 962 ± 137 960 ± 103  959 ± 124  3 1176 ± 108 1003 ± 121 973 ± 105 989 ± 128 1043 ± 158  7 1351 ± 142 1056 ± 151 873 ± 125 890 ± 98 1100 ± 156 10 1591 ± 179 1122 ± 139 722 ± 157 674 ± 96 1172 ± 188 14 1951 ± 225 1417 ± 191 503 ± 151 342 ± 64 1228 ± 174 17 2301 ± 344 1672 ± 262 398 ± 160 216 ± 43 1143 ± 186 21 1794 ± 328 307 ± 169  94 ± 26  996 ± 187 24 1867 ± 408 261 ± 168  62 ± 14  867 ± 178 28 2120 ± 483 217 ± 167  45 ± 16  713 ± 178

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 and ADC in the LU-01-0251 PDX model was calculated based on tumor volume measurements at day 17 after the start of the treatment.

TABLE 34 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Group Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 2301 ± 344 — — — 2 BCY6136, 1672 ± 262 72.7  47.0 p > 0.05 1 mpk, qw 3 BCY6136,  398 ± 160 17.3 142.1 p < 0.001 2 mpk, qw 4 BCY6136,  216 ± 43  9.4 155.6 p < 0.001 3 mpk, qw 5 ADC, 1143 ± 186 49.7  86.3 p < 0.01 3 mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 and ADC in LU-01-0251 PDX model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in the FIG. 15 and Tables 33 and 34.

In this study, the treatment was started when the average tumor volume reached 960 mm³. On day 17 after the start of treatment, the mean tumor volume of vehicle treated mice reached 2301 mm³. BCY6136 at 1 mg/kg qw (TV=1672 mm³, TGI=47.0%, p>0.05) didn't show obvious antitumor activity; BCY6136 at 2 mg/kg qw (TV=398 mm³, TGI=142.1%, p<0.001) and 3 mg/kg qw (TV=216 mm³, TGI=155.6%, p<0.001) produced dose-dependent anti-tumor activity on day 17.

After 70 days' treatment with BCY6136 at 2 mg/kg qw, 3 in 5 of these mice showed complete tumor regression, the other 2 mice showed obvious tumor relapse from day 42 to day 77. Then further treatment with BCY6136 3 mg/kg qw was performed to the two relapse tumors from day 7, one of tumor showed obvious tumor regress while another one showed resistance to the treatment.

After 56 days' treatment with BCY6136 at 3 mg/kg qw, all the mice of this group showed complete tumor regression.

ADC at 3 mg/kg qw (TV=1143 mm³, TGI=86.3%, p<0.01) showed obvious anti-tumor activity on day 17, after another 53 day′ treatment, these mice showed further but not complete tumor regression.

In this study, there were some mice showed sudden bodyweight loss, this may have the relationship with the long term feeding of the immune-deficiency mice.

Study 12: In Vivo Efficacy Study of BCY6033, BCY6136, BCY6082 and BCY6031 in the LU-01-0046 NSCLC PDX Model in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6033, BCY6136, BCY6082 and BCY6031 in large LU-01-0046 PDX tumors in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule BCYs 1 Vehicle 5 — 10 iv qw 2 BCY6082 5 1 10 iv qw 3 BCY6082 5 3 10 iv qw 4 BCY6033 5 1 10 iv qw 5 BCY6033 5 3 10 iv qw 6 BCY6136 5 1 10 iv qw 7 BCY6136 5 3 10 iv qw 8 ADC 5 3 10 iv qw 9 BCY6031 5 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0046 of tumor fragment (˜30 mm³) for tumor development. The treatment was started when the average tumor volume reaches 955 mm³ for BT17BDCs study and 1039 mm³ for BOYs study. The test article administration and the animal numbers in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10% sucrose pH 5 BCY6033 0.1 Dilute 150 μl 1 mg/ml BCY6033 stock with 1350 μl Acetate buffer¹ 0.3 Dilute 450 μl 1 mg/ml BCY6033 stock with 1050 μl Acetate buffer BCY6136 0.1 Dilute 150 μl 1 mg/ml BCY6136 stock with 1350 μl Acetate buffer 0.3 Dilute 450 μl 1 mg/ml BCY6136 stock with 1050 μl Acetate buffer BCY6082 0.1 Dilute 150 μl 1 mg/ml BCY6082 stock with 1350 μl Acetate buffer 0.3 Dilute 450 μl 1 mg/ml BCY6082 stock with 1050 μl Acetate buffer BCY6031 0.3 Dissolve 5.72 mg BCY6031 in 5.6 ml Acetate buffer to make 1 mg/ml stock. Dilute 450 μl 1 mg/ml BCY6031 with 1050 μl Acetate buffer ADC 0.3 Dilute 43 μl 10.47 mg/ml ADC stock solution into 1457 μl with buffer² ¹Acetate buffer: 50 mM Acetate 10% sucrose pH5 ²Dissolve 0.419 g His. hydrochloride in 100 ml water, use 1M HCl adjust PH to 5.5

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 16 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing LU-01-0046 is shown in Table 35.

TABLE 35 Tumor volume trace over time (BCYs Section) Days after the start of treatment Group Treatment 0 4 8 11 15 18 22 1 Vehicle, 1044 ± 115 1762 ± 178 2404 ± 262 — — — — qw 2 BCY6082, 1049 ± 133 1592 ± 178 2279 ± 168 — — — — 1 mpk, qw 3 BCY6082, 1033 ± 111 1040 ± 124 1294 ± 182 1298 ± 101 1849 ± 189 2052 ± 168 1999 ± 425 3 mpk, qw 4 BCY6033, 1030 ± 124 1173 ± 227 1791 ± 324 2408 ± 484 — — — 1 mpk, qw 5 BCY6033, 1046 ± 128  555 ± 85   441 ± 144  182 ± 76   163 ± 94   114 ± 54   88 ± 76  3 mpk, qw 6 BCY6136, 1037 ± 130 1163 ± 146 1927 ± 283 2483 ± 530 — — — 1 mpk, qw 7 BCY6136, 1036 ± 100  784 ± 146  548 ± 107  362 ± 110  325 ± 122  275 ± 152  233 ± 187 3 mpk, qw 8 ADC, 1033 ± 114 1155 ± 230 2200 ± 505 — — — — 3 mpk, qw 9 BCY6031, 1042 ± 117  820 ± 149 1319 ± 233  901 ± 188  672 ± 198  522 ± 315  515 ± 323 3 mpk, qw Note: the tumor volume trace didn't show after the day22 for the group3, 5, 7 and 9.

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the LU-01-0046 PDX model was calculated based on tumor volume measurements at day 22 and day 28 respectively for the two section studies after the start of the treatment.

TABLE 36 Tumor growth inhibition analysis (BCYs section on day 22) Tumor T/C^(b) TGI Group Treatment Volume (%) (%) P value 1 Vehicle, qw 6186 ± 596* — — — 2 BCY6082, 5805 ± 428* 93.8   7.5 p > 0.05 1 mpk, qw 3 BCY6082, 1999 ± 425 32.3  81.2 p < 0.01 3 mpk, qw 4 BCY6033, 4384 ± 881* 70.9  34.8 p > 0.05 1 mpk, qw 5 BCY6033,  88 ± 76  1.4 118.6 P < 0.001 3 mpk, qw 6 BCY6136, 4564 ± 981* 73.8  31.4 p > 0.05 1 mpk, qw 7 BCY6136,  233 ± 187  3.8 115.6 p < 0.001 3 mpk, qw 8 ADC, 5446 ± 1250* 88.0  14.2 p > 0.05 3 mpk, qw 9 BCY6031,  515 ± 323  8.3 110.2 p < 0.001 3 mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividing the average tumor volume of the treated group by the average tumor volume of the control group (T/C). *Some groups was terminated before day 22, and the tumor size was calculated by exponential growth equation acquisition as below: Vehicle group: Y = 995.4 × exp (0.1134 × X). BCY6082, 1 mpk group: Y = 939.1 × exp (0.1128 × X). BCY6033, 1 mpk group: Y = 846.6 × exp (0.0945 × X). BCY6136, 1 mpk group: Y = 855.0 × exp (0.0974 × X). ADC, 3 mpk group: Y = 757.4 × exp (0.1312 × X).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in large LU-01-0046 tumors was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIG. 16 and Tables 35 and 36.

In BCYs study, the mean tumor size of vehicle treated mice was calculated as 6186 mm³ on day 22. BCY6082, BCY6033, BCY6136 at 1 mg/kg and ADC at 3 mg/kg didn't show obvious anti-tumor activity when starting treatment from tumor size of 1000 mm³.

BCY6082 (TV=1999 mm³, TGI=81.2%, p<0.01), BCY6033 (TV=88 mm³, TGI=118.6%, p<0.001), BCY6136 (TV=233 mm³, TGI=115.6%, p<0.001) and BCY6031 (TV=115 mm³, TGI=110.2%, p<0.001) at 3 mg/kg produced significant anti-tumor antitumor activity. Among them, BCY6033 and BCY6136 eradicated 2/5 and 4/5 tumors completely.

Study 13: In Vivo Efficacy of BCY6136 in Balb/c Nude Mice Bearing LU-01-0046 NSCLC PDX Model

(a) Study Objective

The objective of the research was to evaluate the in vivo therapeutic efficacy of BCY6136 in Balb/c nude mice bearing LU-01-0046 NSCLC PDX model.

(b) Experimental Design

Dose Dosing Group Treatment n (mg/kg) Route Schedule 1 Vehicle 5 — i.v. qw * 2 w 2 BCY6136 5 1 i.v. qw * 3 w 3 BCY6136 5 2 i.v. qw * 4 w 4 BCY6136 5 3 i.v. qw * 4 w 5 ADC 5 3 i.v. qw * 3 w 6 ADC 5 5 i.v. qw * 3 w Note: Groups were terminated when average tumor volume reached over 2000 mm³ and tumors were harvested for FFPE: Group 1 on PG-D14, group 5 on PG-D18, group 2 & 6 on PG-D21 and group 3 & 4 on PG-D31.

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with certain kind of tumor fragment (˜30 mm³) for tumor development. The treatments were started when the average tumor volume reached approximately 198 mm³. The test article administration and the animal numbers in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Com- Dose Con. Gr pounds (mg/kg) (mg/ml) Formulation 1 Vehicle — — 50 mM Acetate, 10% Sucrose pH 5 (without DMSO) 2 BCY6136 1 0.1 Dissolve 10.93 mg BCY6136 in 10.766 ml vehicle, ultrasonic simply to make the 1 mg/ml BCY6136 stock solution Dilute 150 μl 1 mg/ml BCY6136 stock solution with 1350 μl vehicle 3 BCY6136 2 0.2 Dilute 300 μl 1 mg/ml BCY6136 stock solution with 1200 μl vehicle 4 BCY6136 3 0.3 Dilute 450 μl 1 mg/ml BCY6136 stock solution with 1050 μl vehicle Buffer 2: Dissolve 0.419 g His. hydrochloride in 100 ml water, use 1M HCl adjust pH to 5.5 5 ADC 3 0.3 Dilute 43 μl 10.47 mg/ml ADC stock solution with 1457 μl with buffer 2 6 ADC 5 0.5 Dilute 71.6 μl 10.47 mg/ml ADC stock solution with 1428.4 μl with buffer 2 Note The dosing formulation frequently is fresh prepared timely.

(iii) Sample Collection

Groups were terminated when average tumor volume reached over 2000 mm³ and tumors were harvested for FFPE after the last measurement: Group 1 on PG-D14, group 5 on PG-D18, group 2 & 6 on PG-D21 and group 3 & 4 on PG-D31.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 17 .

(ii) Tumor Volume Trace

Mean tumor volume overtime in female Balb/c nude mice bearing LU-01-0046 NSCLC PDX model is shown in Table 37.

TABLE 37 Tumor volume trace over time (mm³) 2 3 4 5 6 1 BCY6136 BCY6136 BCY6136 ADC ADC Gr Vehicle 1 mpk, 2 mpk, 3 mpk, 3 mpk, 5 mpk, Treatment qw qw qw qw qw qw 0  201 ± 37   198 ± 39  201 ± 40  200 ± 46   195 ± 28   195 ± 40  3  441 ± 82   310 ± 59  283 ± 77  155 ± 40   418 ± 99   389 ± 68  7  927 ± 171  547 ± 88  423 ± 132  74 ± 19   643 ± 159  596 ± 116 10 1546 ± 377  747 ± 121 321 ± 108  31 ± 8    938 ± 230  882 ± 134 14 2307 ± 594 1058 ± 140 264 ± 95   26 ± 11  1475 ± 466 1215 ± 193 17 — 1390 ± 205 127 ± 41   26 ± 13  2281 ± 556 1576 ± 228 21 — 2138 ± 301 118 ± 34   64 ± 42  — 2049 ± 242 24 — — 101 ± 40   99 ± 63  — — 28 — — 255 ± 140 276 ± 176 — — 31 — — 582 ± 346 477 ± 283 — —

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in Balb/c nude mice bearing LU-01-0046 PDX model was calculated based on tumor volume measured on PG-D14.

TABLE 38 Tumor growth inhibition analysis P value Tumor compared Volume T/C TGI with Gr Treatment (mm³)^(a) (%)^(b) (%)^(c) vehicle 1 Vehicle qw 2307 ± 594 — — — 2 BCY6136 1058 ± 140 45.9  59.1 p < 0.05 1 mpk, qw 3 BCY6136  264 ± 95 11.4  97.0 p < 0.001 2 mpk, qw 4 BCY6136  26 ± 11  1.1 108.3 p < 0.001 3 mpk, qw 5 ADC 1475 ± 466 63.9  39.2 p > 0.05 3 mpk, qw 6 ADC 1215 ± 193 52.7  51.6 p > 0.05 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition was calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). ^(c)TGI was calculated for each group using the formula: TGI (%) = [1 − (T_(i) − T₀)/(V_(i) − V₀)] × 100

(e) Results Summary and Discussion

In the present study, the therapeutic efficacy of test articles in the LU-01-0046 PDX model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points were shown in the FIG. 17 and Tables 37 and 38.

The mean tumor size of vehicle treated mice reached 2307 mm³ on PG-D14. BCY6136 at 1 mg/kg (TV=1058 mm³, TGI=59.1%, p<0.05), at 2 mg/kg (TV=264 mm³, TGI=97.0%, p<0.001) and at 3 mg/kg (TV=26 mm³, TGI=108.3%, p<0.001) produced dose-dependent antitumor activity. ADC at 3 mg/kg and 5 mg/kg did not show obvious antitumor activity (p>0.05).

In this study, all of the group's animals maintained the body weight well.

Study 14: In Vivo Efficacy Study of BCY6033, BCY6136, BCY6082, BCY6173, BCY6175 and BCY6031 in the LU-01-0046 NSCLC PDX Model in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of test articles in the LU-01-0046 NSCLC PDX model in Balb/c nude mice.

(b) Experimental Design

Dose Dosing Dosing Group Treatment n (mg/kg) Volume (μl/g) Route Schedule Part 1 1 Vehicle 5 — 10 iv qw 2 BCY6033 5 ½ 10 iv qw 3 BCY6033 5 3 10 iv qw 4 BCY6136 5 ½ 10 iv qw 5 BCY6136 5 3 10 iv qw 6 BCY6082 5 1 10 iv qw 7 BCY6082 5 3 10 iv qw Part 2 8 Vehicle 5 — 10 iv qw 9 BCY6173 5 1 10 iv qw 10 BCY6173 5 3 10 iv qw 11 BCY6175 5 3 10 iv qw 12 BCY6031 5 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0046 of tumor fragment (˜30 mm³) for tumor development. The treatment was started when the average tumor volume reaches 200 mm³ for part 1 study and 192 mm³ for part 2 study. The test article administration and the animal numbers in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10% sucrose pH 5 BCY6033 0.1 Dilute 150 μl 1 mg/ml BCY6033 stock with 1350 μl Acetate buffer¹ 0.3 Dilute 450 μl 1 mg/ml BCY6033 stock with 1050 μl Acetate buffer BCY6136 0.1 Dilute 150 μl 1 mg/ml BCY6136 stock with 1350 μl Acetate buffer 0.3 Dilute 450 μl 1 mg/ml BCY6136 stock with 1050 μl Acetate buffer BCY6082 0.1 Dilute 150 μl 1 mg/ml BCY6082 stock with 1350 μl Acetate buffer 0.3 Dilute 450 μl 1 mg/ml BCY6082 stock with 1050 μl Acetate buffer BCY6173 0.1 Dissolve 3.65 mg BCY6173 in 3.5 ml Acetate buffer to make 1 mg/ml stock. Dilute 150 μl 1 mg/ml BCY6173 with 1350 μl Acetate buffer 0.3 Dilute 450 μl 1 mg/ml BCY6173 stock with 1050 μl Acetate buffer BCY6175 0.3 Dissolve 3.02 mg BCY6175 in 2.9 ml Acetate buffer to make 1 mg/ml stock. Dilute 450 μl 1 mg/ml BCY6175 with 1050 μl Acetate buffer BCY6031 0.3 Dissolve 5.72 mg BCY6031 in 5.6 ml Acetate buffer to make 1 mg/ml stock. Dilute 450 μl 1 mg/ml BCY6031 with 1050 μl Acetate buffer ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIGS. 18 to 22 .

(ii) Tumor Volume Trace

Mean tumor volume on day 21 after the start of treatment in female Balb/c nude mice bearing LU-01-0046 is shown in Tables 39 and 40.

TABLE 39 Tumor volume trace over time (Part 1) Days after the start of treatment Gr Treatment 0 3 6 10 14 17 21 1 Vehicle, 202 ± 26 328 ± 48 536 ± 68 953 ± 107 1386 ± 97  1833 ± 132 2551 ± 242 qw 2 BCY6033, 201 ± 23 285 ± 47 449 ± 87 623 ± 112  891 ± 196  967 ± 228 1285 ± 234 1 mpk, qw 3 BCY6033, 201 ± 26 187 ± 43  91 ± 34  37 ± 14    3 ± 3      0 ± 0      0 ± 0   3 mpk, qw 4 BCY6136, 200 ± 33 293 ± 56 426 ± 91 682 ± 151  964 ± 194  976 ± 258 1285 ± 234 1 mpk, qw 5 BCY6136, 201 ± 33 194 ± 31 135 ± 27  52 ± 18   13 ± 9      4 ± 4      0 ± 0   3 mpk, qw 6 BCY6082, 201 ± 29 295 ± 43 466 ± 65 877 ± 80 1201 ± 106 1502 ± 108 1826 ± 224 1 mpk, qw 7 BCY6082, 201 ± 34 235 ± 36 310 ± 44 398 ± 65  634 ± 136  729 ± 184 1042 ± 290 3 mpk, qw

TABLE 40 Tumor volume trace over time (Part 2) Days after the start of treatment Gr Treatment 0 3 7 10 14 17 21 8 Vehicle, 192 ± 30 311 ± 83 562 ± 146 830 ± 230 1320 ± 444 1652 ± 528 2342 ± 651 qw 9 BCY6173, 191 ± 33 318 ± 58 553 ± 88  817 ± 165 1314 ± 276 1546 ± 276 2151 ± 262 1 mpk, qw 10 BCY6173, 192 ± 37 259 ± 51 400 ± 53  455 ± 28   636 ± 92   646 ± 138  890 ± 260 3 mpk, qw 11 BCY6175, 192 ± 42 186 ± 57  92 ± 38   19 ± 11     0 ± 0      0 ± 0      0 ± 0   3 mpk, qw 12 BCY6031, 191 ± 38 207 ± 46 387 ± 70  355 ± 110  544 ± 159  643 ± 185  874 ± 281 3 mpk, qw

(ii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the LU-01-0046 PDX model was calculated based on tumor volume measurements at day 21 after the start of the treatment.

TABLE 41 Tumor growth inhibition analysis (Part 1) Tumor T/C^(b) TGI Group Treatment Volume (%) (%) P value 1 Vehicle, qw 2551 ± 242 — — — 2 BCY6033, 1285 ± 234 50.4  53.9 p < 0.001 1 mpk, qw 3 BCY6033,   0 ± 0  0.0 108.6 p < 0.001 3 mpk, qw 4 BCY6136, 1285 ± 234 50.4  53.9 p < 0.001 1 mpk, qw 5 BCY6136,   0 ± 0  0.0 108.5 p < 0.001 3 mpk, qw 6 BCY6082, 1826 ± 224 71.6  30.8 p < 0.05 1 mpk, qw 7 BCY6082, 1042 ± 290 40.8  64.2 p < 0.001 3 mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

TABLE 42 Tumor growth inhibition analysis (Part 2) Tumor T/C^(b) TGI Group Treatment Volume (%) (%) P value  8 Vehicle, qw 2342 ± 651 — — —  9 BCY6173, 2151 ± 262 91.8   8.9 p > 0.05 1 mpk, qw 10 BCY6173,  890 ± 260 38.0  67.5 p < 0.05 3 mpk, qw 11 BCY6175,   0 ± 0  0.0 108.9 p < 0.001 3 mpk, qw 12 BCY6031,  874 ± 281 37.3  68.2 p < 0.05 3 mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the LU-01-0046 PDX model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIGS. 18 to 22 and Tables 39 to 42.

In part 1 study, the mean tumor size of vehicle treated mice reached 2551 mm³ on day 21 after the start of treatment.

BCY6033 at ½ mg/kg, qw (TV=1285 mm³, TGI=53.9%, p<0.001) and BCY6136 at ½ mg/kg, qw (TV=1285 mm³, TGI=53.9%, p<0.001) produced significant anti-tumor activity, but didn't exhibit any tumor regression. BCY6033 at 3 mg/kg, qw (TV=0 mm³, TGI=108.6%, p<0.001) and BCY6136 at 3 mg/kg, qw (TV=0 mm³, TGI=108.5%, p<0.001) completely eradicated the tumors, 1 of 5 tumors respectively in BCY6033 and BCY6136 3 mg/kg groups showed regrowth after the dosing suspension and the tumors were resistant to BCY6033 or BICY6136 treatment when resuming the dosing. The remaining tumors in the BCY6033 and BCY6136 groups (4/5 for each group) showed no regrowth after 80 days of dosing suspension. BCY6082 at 1 mg/kg, qw (TV=1826 mm³, TGI=30.8%, p<0.05) and 3 mg/kg, qw (TV=1042 mm³, TGI=64.2%, p<0.001) produced dose-dependent anti-tumor activity, but didn't show tumor regression.

In part 2 study, the mean tumor size of vehicle treated mice reached 2342 mm³ on day 21 after the start of treatment. BCY6173 at 1 mg/kg, qw (TV=2151 mm³, TGI=8.9%, p>0.05) did not show anti-tumor antitumor activity. BCY6173 at 3 mg/kg, qw (TV=890 mm³, TGI=67.5%, p<0.05) produced obvious anti-tumor activity.

BCY6175 at 3 mg/kg, qw (TV=0 mm³, TGI=108.9%, p<0.001) completely eradicated 4/5 tumors on day 14. BCY6031 at 3 mg/kg, qw (TV=874 mm³, TGI=68.2%, p<0.05) produced obvious anti-tumor activity, but didn't show any tumor regression.

Study 15: In Vivo Efficacy Study of BCY6136 in the LU-01-0412 NSCLC PDX Model in Balb/c Nude Mice

(a) Study Objective

The objective of the project is to evaluate the in vivo therapeutic efficacy of BCY6136 in the LU-01-0412 NSCLC PDX model in BALB/c nude mice.

(b) Experimental Design

Dose Dosing Dosing Gr Treatment n (mg/kg) Volume (μl/g) Route Schedule 1 Vehicle 6 — 10 iv Qw, 4 2 BCY6136 6 1 10 iv Qw, 4 3 BCY6136 6 3 10 iv Qw, 4 4 BCY8245 6 3 10 iv Qw, 4 5 BCY8781 6 3 10 iv Qw, 4

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0412 tumor fragment (˜30 mm³) for tumor development. Animals were randomized when the average tumor volume reached 159 mm³. The test article administration and the animal numbers in each group were shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 25 mM Histidine 10% sucrose pH 7 BCY6136 1 Dissolve 6.06 mg BCY6136 in 5.969 ml 50 mM Acetate/ acetic acid pH 5 10% sucrose 0.1 Dilute 180 μl 1 mg/ml BT5528 with 1620 μl 50 mM Acetate/acetic acid pH 5 10% sucrose 0.3 Dilute 540 μl 1 mg/ml BT5528 with 1260 ul 50 mM Acetate/acetic acid pH 5 10% sucrose BCY8245 1 Dissolve 4.15 mg BCY8245 powder in 4.121 ml vehicle buffer 0.3 Dilute 540 μl 1 mg/ml BCY8245 with 1260 μl vehicle buffer BCY8781 1 Dissolve 4.08 mg BCY8781 powder in 80.8 μl DMSO, then dilute to 1 mg/ml with 3.958 vehicle buffer 0.3 Dilute 540 μl 1 mg/ml BCY8781 with 1260 μl vehicle buffer

(iii) Sample Collection

Plasma from vehicle and 3 extra mice treated with BCY6136, BCY8245 and BCY8781 were collected at 30 min and 24 h post dosing. Tumor from vehicle and 3 extra mice treated with BCY6136, BCY8245 and BCY8781 were collected at 24 h post dosing.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curves are shown in FIG. 23 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female BALB/c nude mice bearing LU-01-0412 xenograft is shown in Table 43.

TABLE 43 Tumor volume trace over time Group 1 Group 2 Group 3 Group 4 Group 5 Vehicle BCY6136 BCY6136 BCY8245 BCY8781 Days Qw*4 1 mpk, Qw*4 3 mpk, Qw*4 3 mpk, Qw*4 3 mpk, Qw*4  0  159 ± 11 159 ± 13 159 ± 11 159 ± 12 159 ± 11  4  255 ± 12 214 ± 16 197 ± 16 168 ± 18 176 ± 21  7  309 ± 20 237 ± 16 195 ± 16 132 ± 10 167 ± 13 11  395 ± 31 246 ± 19 156 ± 18  78 ± 4 107 ± 15 14  464 ± 31 300 ± 18 177 ± 29  45 ± 5  72 ± 12 18  521 ± 26 369 ± 32 210 ± 32  21 ± 2  44 ± 8 21  611 ± 33 470 ± 46 225 ± 32  11 ± 1  31 ± 6 25  737 ± 68 632 ± 47 252 ± 37  6 ± 1  20 ± 6 28  788 ± 80 664 ± 52 299 ± 37  2 ± 1  14 ± 5 32 1104 ± 142 758 ± 70 416 ± 52  1 ± 1  12 ± 5

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136, BCY8245 and BCY8781 in the LU-01-0412 xenograft model was calculated based on tumor volume measurements on day 32 after the start of the treatment.

TABLE 44 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Group Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, 1104 ± 142 — — — qw * 4 2 BCY6136,  758 ± 70 68.6  36.7 p < 0.05 1 mpk, qw * 4 3 BCY6136,  416 ± 52 37.6  72.9 p < 0.001 3 mpk, qw * 4 4 BCY8245,   1 ± 1  0.1 116.8 p < 0.001 3 mpk, qw * 4 5 BCY8781,  12 ± 5  1.0 115.6 p < 0.001 3 mpk, qw * 4 ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136, BCY8245 and BCY8781 in the LU-01-0412 xenograft model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in FIG. 23 and Tables 43 and 44.

The mean tumor volume of vehicle treated mice reached 1104 mm³ on day 32 after the start of treatment. BCY6136 at 1 mg/kg, qw*4 (TV=758 mm³, TGI=36.7%, p<0.05) and 3 mg/kg, qw*4 (TV=416 mm³, TGI=72.9%, p<0.001) produced dose-dependent antitumor activity, but didn't show any tumor regression. BCY8245 at 3 mg/kg, qw*4 (TV=1 mm³, TGI=116.8%, p<0.001) and BCY8781 at 3 mg/kg, qw*4 (TV=12 mm³, TGI=115.6%, p<0.001) regressed the tumors obviously. Among them, 5 of 6 tumor treated with BCY8245 3 mg/kg and 2 of 6 tumor treated with d BCY8781 3 mg/kg were completely eradicated on day 32.

In this study, animals in all groups maintained the body weight well.

Study 16: In Vivo Efficacy Study of BCY6136 in Treatment of LU-01-0486 PDX Model in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in the LU-01-0486 PDX model in Balb/c nude mice.

(b) Experimental Design

Dose Dosing Dosing Gr Treatment n (mg/kg) Volume (μl/g) Route Schedule 1 Vehicle 5 — 10 iv qw 2 BCY6136 5 1 10 iv qw 3 BCY6136 5 2 10 iv qw 4 BCY6136 5 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0486 of tumor fragment (˜30 mm³) for tumor development. The treatment was started when the average tumor volume reached 180 mm³ for efficacy study. The test article administration and the animal number in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10% sucrose pH 5 BCY6136 0.3 0.3 mg/ml BCY6136 was prepared as described in Study 10 0.2 Dilute 940 μl 0.3 mg/ml BCY6136 with 470 μl Acetate buffer¹ 0.1 Dilute 470 μl 0.3 mg/ml BCY6136 with 940 μl Acetate buffer ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 24 .

(ii) Tumor Volume Trace

Mean tumor volume on day 14 after the start of treatment in female Balb/c nude mice bearing LU-01-0486 xenograft is shown in Table 45.

TABLE 45 Tumor volume trace over time Days after the start of treatment Group Treatment 0 3 7 10 14 1 Vehicle, 179 ± 20 232 ± 30 358 ± 45 450 ± 47 651 ± 112 qw 2 BCY6136, 180 ± 23 221 ± 20 326 ± 34 420 ± 34 638 ± 71  1 mpk, qw 3 BCY6136, 179 ± 27 222 ± 26 365 ± 44 459 ± 82 645 ± 105 2 mpk, qw 4 BCY6136, 180 ± 25 209 ± 37 304 ± 51 348 ± 77 449 ± 115 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the LU-01-0486 PDX model was calculated based on tumor volume measurement at day 14 after the start of the treatment.

TABLE 46 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Group Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 651 ± 112 — — — 2 BCY6136, 638 ± 71  98.0 3.0 p > 0.05 1 mpk, qw 3 BCY6136, 645 ± 105 99.1 1.2 p > 0.05 2 mpk, qw 4 BCY6136, 449 ± 115 68.9 43.1 p > 0.05 3 mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in LU-01-0486 PDX model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in the FIG. 24 and Tables 45 and 46.

In this study, the mean tumor volume of vehicle treated mice reached 651 mm³ on day 14 after the start of treatment. BCY6136 at 1 mg/kg, qw (TV=638 mm³, TGI=3.0%, p>0.05) and 2 mg/kg, qw (TV=645 mm³, TGI=1.2%, p>0.05) didn't show any anti-tumor activity. BCY6136 at 3 mg/kg, qw (TV=449 mm³, TGI=43.1%, p>0.05) produced slight anti-tumor activity without statistical significance.

Study 17: In Vivo Efficacy Test of BCY6033, BCY6136 and BCY6082 in Treatment of MDA-MB-231-Luc Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCY6033, BCY6136 and BCY6082 in treatment of MDA-MB-231-luc xenograft model in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Gr Treatment n (mg/kg) (μl/g) Route Schedule 1 Vehicle 3 — 10 iv qw 2 BCY6033 3 1 10 iv qw 3 BCY6033 3 2 10 iv qw 4 BCY6033 3 3 10 iv qw 5 BCY6136 3 1 10 iv qw 6 BCY6136 3 2 10 iv qw 7 BCY6136 3 2 10 iv qw 8 BCY6082 3 2 10 iv qw 9 BCY6082 3 5 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with MDA-MB-231-luc tumor cells (10×10{circumflex over ( )}6) in 0.1 ml of PBS with 0.1 ml matrigel for tumor development. 36 animals were randomized when the average tumor volume reached 159 mm³. The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Dose Treatment (mg/ml) Formulation Vehicle 50 mM Acetate, 10% sucrose pH = 5 BCY6033 1 Dissolve 6.71 mg BCY6033 into 6.710 ml formulation buffer 0.3 Dilute 270 μl 1 mg/ml BCY6033 into 630 μl formulation buffer 0.2 Dilute 180 μl 1 mg/ml BCY6033 into 720 μl formulation buffer 0.1 Dilute 90 μl 1 mg/ml BCY6033 into 810 μl formulation buffer BCY6136 1 Dissolve 3.79 mg BCY6136 into 3.695 ml formulation buffer 0.3 Dilute 270 μl 1 mg/ml BCY6136 into 630 μl formulation buffer 0.2 Dilute 180 μl 1 mg/ml BCY6136 into 720 μl formulation buffer 0.1 Dilute 90 μl 1 mg/ml BCY6136 into 810 μl formulation buffer BCY6082 1 Weigh and dissolve 4.30 mg BCY6082 into 4.162 ml formulation buffer 0.5 Dilute 450 μl 1 mg/ml BCY6082 into 450 μl formulation buffer 0.2* Dilute 180 μl 1 mg/ml BCY6082 into 720 μl formulation buffer

(iv) Sample Collection

On PG-D24, we collected and fixed the tumors of Group 1, 8 and 9 for FFPE.

On PG-D33, we collected and fixed the tumors of Group 2 and 5 for FFPE.

At the end of study, we collected and fixed the tumors of Group 3, 4, 6 and 7 for FFPE.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth are shown in FIGS. 25 to 27 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing MDA-MB-231-luc xenograft is shown in Tables 47 to 49.

TABLE 47 Tumor volume trace (PG-D0~PG-D17) Days after the start of treatment Gr. Treatment 0 2 4 7 9 11 14 17 1 Vehicle, 159 ± 14 269 ± 8  306 ± 19 425 ± 52 688 ± 54 908 ± 54 1064 ± 98 1315 ± 95 qw 2 BCY6033, 159 ± 6  219 ± 19 221 ± 55 296 ± 76 329 ± 64 421 ± 77 479 ± 84   609 ± 122 1 mpk, qw 3 BCY6033, 159 ± 10 240 ± 73 215 ± 57 201 ± 47 109 ± 36  84 ± 34  64 ± 32    59 ± 35  2 mpk, qw 4 BCY6033, 158 ± 7  189 ± 27 147 ± 32 109 ± 26  79 ± 11  66 ± 7   41 ± 5     31 ± 6   3 mpk, qw 5 BCY6136, 159 ± 10 226 ± 36 221 ± 54 310 ± 72 416 ± 89 526 ± 77 636 ± 92   809 ± 135 1 mpk, qw 6 BCY6136, 159 ± 16 218 ± 17 182 ± 22 182 ± 26 101 ± 20  77 ± 24  36 ± 4     41 ± 10  2 mpk, qw 7 BCY6136, 158 ± 5  241 ± 12 259 ± 6  325 ± 14 258 ± 12 246 ± 15 162 ± 19   178 ± 10  3 mpk, qw 8 BCY6082, 159 ± 13 210 ± 10 242 ± 16 305 ± 19 445 ± 58 611 ± 76 734 ± 139  926 ± 105 2 mpk, qw 9 BCY6082, 159 ± 7  227 ± 31 247 ± 47 250 ± 65 276 ± 79 241 ± 61 220 ± 56   184 ± 85  5 mpk, qw

TABLE 48 Tumor volume trace (PG-D19~PG-D33) Days after the start of treatment Gr. Treatment 19 21 24 26 28 31 33 1 Vehicle, qw 1453 ± 128  1661 ± 173  — — — — — 2 BCY6033, 724 ± 162 880 ± 156 1069 ± 189  1182 ± 164  1342 ± 166  1647 ± 113  — 1 mpk, qw 3 BCY6033, 61 ± 35 67 ± 44 100 ± 76  133 ± 96  163 ± 106 221 ± 143 257 ± 152 2 mpk, qw 4 BCY6033, 29 ± 7  22 ± 12 22 ± 8  21 ± 9  21 ± 10 43 ± 20 57 ± 29 3 mpk, qw 5 BCY6136, 879 ± 190 994 ± 213 1253 ± 313  1431 ± 353  1507 ± 253  2181 ± 609  — 1 mpk, qw 6 BCY6136, 35 ± 9  33 ± 9  31 ± 17 41 ± 32 59 ± 45 82 ± 59 87 ± 71 2 mpk, qw 7 BCY6136, 171 ± 21  132 ± 19  108 ± 19  85 ± 15 81 ± 8  87 ± 14 92 ± 18 3 mpk, qw 8 BCY6082, 1034 ± 178  1287 ± 94  — — — — — 2 mpk, qw 9 BCY6082, 214 ± 120 218 ± 146 — — — — — 5 mpk, qw

TABLE 49 Tumor volume trace (PG-D35~PG-D47) Days after the start of treatment Gr. Treatment 35 38 40 42 45 47 3 BCY6033, 352 ± 210 456 ± 271 525 ± 302 683 ± 400 738 ± 429 853 ± 476 2 mpk, qw 4 BCY6033, 79 ± 47 118 ± 71  139 ± 82  220 ± 125 312 ± 176 423 ± 222 3 mpk, qw 6 BCY6136, 124 ± 106 156 ± 120 179 ± 142 239 ± 197 285 ± 239 350 ± 298 2 mpk, qw 7 BCY6136, 129 ± 38  173 ± 65  181 ± 65  269 ± 113 293 ± 114 371 ± 128 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6033, BCY6136 and BCY6082 in the MDA-MB-231-luc xenograft model was calculated based on tumor volume measurements at day 21 after the start of treatment.

TABLE 50 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Gr Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 1661 ± 173 — — — 2 BCY6033,  880 ± 156 53.0  52.0 p < 0.001 1 mpk, qw 3 BCY6033,  67 ± 44  4.1 106.1 p < 0.001 2 mpk, qw 4 BCY6033,  22 ± 12  1.3 109.1 p < 0.001 3 mpk, qw 5 BCY6136,  994 ± 213 59.8  44.4 p < 0.01 1 mpk, qw 6 BCY6136,  33 ± 9  2.0 108.4 p < 0.001 2 mpk, qw 7 BCY6136,  132 ± 19  8.0 101.7 p < 0.001 3 mpk, qw 8 BCY6082, 1287 ± 94 77.5  24.9 p > 0.05 2 mpk, qw 9 BCY6082,  218 ± 146 13.1  96.1 p < 0.001 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6033, BCY6136 and BCY6082 in the MDA-MB-231-luc xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIGS. 25 to 27 and Tables 47 to 50.

The mean tumor size of vehicle treated mice reached 1661 mm³ on day 21. BCY6033 at 1 mg/kg (TV=880 mm³, TGI=52.0%, p<0.001), 2 mg/kg (TV=67 mm³, TGI=106.1%, p<0.001) and 3 mg/kg (TV=22 mm³, TGI=109.1%, p<0.001) produced dose-dependent antitumor activity. BCY6033 at 2 mg/kg and 3 mg/kg regressed the tumors potently, but the tumors showed obvious re-growth from day 21.

BCY6136 at 1 mg/kg (TV=994 mm³, TGI=44.4%, p<0.01) showed moderate antitumor activity, BCY6136 at 2 mg/kg (TV=33 mm³, TGI=108.4%, p<0.001) and 3 mg/kg (TV=132 mm³, TGI=101.1%, p<0.001) produced potent antitumor activity, but the tumors showed obvious re-growth from day 28.

BCY6082 at 2 mg/kg (TV=1287 mm3, TGI=24.9%, p>0.05) didn't show obvious antitumor activity, BCY6082 at 5 mg/kg (TV=218 mm3, TGI=96.1%, p<0.001) produced significant antitumor activity.

In this study, one mouse treated with BCY6136 2 mg/kg lost over 15% bodyweight during the treatment schedule, other mice maintained the bodyweight well.

Study 18: In Vivo Efficacy Test of BCY6136 in Treatment of EMT-6 Syngeneic Model in BALB/c Mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of EMT-6 syngeneic model in BALB/c mice.

(b) Experimental Design

Dose Dosing Group Treatment (mg/kg) N Route Schedule Sample Collection 1 Vehicle — 5 iv qw*4 tumors from spare 2 BCY6136 3 5 iv qw*4 mice will be 3 BCY6136 1/5^(b) 5 iv qw*4 collected for 4 BCY6136 0.3/3^(b) 5 iv qw*4 FACS ^(a)The injection volume of each mouse is 10 ml/kg. ^(b)The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from Day 14.

(c) Experimental Methods and Procedures

(i) Cell Culture

The EMT-6 tumor cells were maintained in vitro as a monolayer culture in EMEM medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with EMT-6 tumor cells (5×10⁶) in 0.1 ml of PBS for tumor development. 44 animals were randomized when the average tumor volume reached 75 mm³. The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

BCY6136 formulation Treatment Conc.(mg/ml) Formulation Vehicle/ — 50 mM Acetate, 10% sucrose buffer pH = 5 BCY6136 1 Dissolve 6.2 mg BCY6136 with 6113 ul buffer BCY6136 0.3 Dilute 450 μl 1 mg/ml BCY6136 stock with 1050 μl buffer BCY6136 0.1 Dilute 150 μl 1 mg/ml BCY6136 stock with 1350 μl buffer BCY6136 0.03 Dilute 45 μl 1 mg/ml BCY6136 stock with 1455 μl buffer

BCY6136 formulation Treatment Conc.(mg/ml) Formulation Vehicle/ — 50 mM Acetate, 10% sucrose buffer pH = 5 BCY6136 1 stock BCY6136 0.3 Dilute 420 μl 1 mg/ml BCY6136 stock with 980 μl buffer BCY6136 0.3 Dilute 420 μl 1 mg/ml BCY6136 stock with 980 μl buffer BCY6136 0.5 Dilute 700 μl 1 mg/ml BCY6136 stock with 700 μl buffer

(iv) Sample Collection

3 tumors from spare mice were collected for FACS on day 11. The data was supplied by biology team.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 28 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female BALB/c mice bearing EMT-6 syngeneic is shown in Table 51.

TABLE 51 Tumor volume trace over time Days after the start of treatment Gr. Treatment 0 3 5 7 10 12 14 17 19 21 1 Vehicle, qw 82 ± 4  141 ± 11  260 ± 24  443 ± 90  557 ± 99  703 ± 119 812 ± 139 948 ± 191 1129 ± 248  1499 ± 340  2 BCY6136, 82 ± 4  58 ± 1  59 ± 2  125 ± 18  240 ± 23  322 ± 23  374 ± 22  431 ± 37  486 ± 50  561 ± 61  3 mpk, qw 3 BCY6136, 82 ± 4  108 ± 18  204 ± 27  350 ± 57  426 ± 49  588 ± 72  691 ± 65  850 ± 98  1018 ± 115  1272 ± 140  1/5^(a) mpk, qw 4 BCY6136, 82 ± 4  130 ± 16  255 ± 35  358 ± 34  450 ± 67  607 ± 94  731 ± 112 872 ± 119 1082 ± 133  1394 ± 161  0.3/3^(a) mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in EMT-6 syngeneic model was calculated based on tumor volume measurements on day 21 after the start of treatment.

TABLE 52 Tumor growth inhibition analysis Tumor P value Volume T/C^(b) TGI compare Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw 1499 ± 340 — — — 2 BCY6136,  561 ± 61 37.4 66.2 p < 0.05 3 mpk, qw 3 BCY6136, 1272 ± 140 84.8 16.1 ns 1/5^(c) mpk, qw 4 BCY6136, 1394 ± 161 93.0  7.4 ns 0.3/3^(c) mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). ^(c)The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from Day 14.

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in EMT-6 syngeneic model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIG. 28 and Tables 51 and 52.

The mean tumor size of vehicle treated mice reached 1499 mm³ on day 21. BCY6136 at 3 mg/kg, qw (TV=561 mm³, TGI=66.2%, p<0.05) showed obvious antitumor activity. BCY6136 at ⅕ mg/kg, qw (TV=1272 mm³, TGI=16.1%, p>0.05) and BCY6136 at 0.3/3 mg/kg, qw (TV=1394 mm³, TGI=7.4%, p>0.05) didn't show any antitumor activity.

The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from day 14. Tumor ulceration was found in mouse 3-5 on Day 14, and the mice was deal with antibiotic cream. In this study, all mice maintained the bodyweight well.

Study 19: In Vivo Efficacy Study of BCY6136 in Treatment of NCI-N87 Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of NCI-N87 xenograft in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule 1 Vehicle 3 — 10 iv Qw 2 BCY6136 3 1 10 iv Qw 3 BCY6136 3 2 10 iv Qw 4 BCY6136 3 3 10 iv Qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The NCI-N87 tumor cells were maintained in RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with NCI-N87 tumor cells (10×10⁶) with matrigel (1:1) in 0.2 ml of PBS for tumor development. The animals were randomized and treatment was started when the average tumor volume reached approximately 176 mm³. The test article administration and the animal number in each group are shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10% sucrose pH 5 BCY6136 1 Dissolve 4.295 mg BCY6136 in 4.214 ml Acetate buffer¹ 0.1 Dilute 90 μl 1 mg/ml BCY6136 stock with 810 μl Acetate buffer 0.2 Dilute 180 μl 1 mg/ml BCY6136 stock with 720 μl Acetate buffer 0.3 Dilute 270 μl 1 mg/ml BCY6136 stock with 630 μl Acetate buffer ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve is shown in FIG. 29 .

(ii) Tumor Volume Trace

Mean tumor volume overtime in female Balb/c nude mice bearing NCI-N87 xenograft is shown in Table 53.

TABLE 53 Tumor volume trace over time Days after the start of treatment Gr. Treatment 0 2 4 7 9 11 14 1 Vehicle, qw 174 ± 7  213 ± 5  266 ± 6  421 ± 10  537 ± 17  598 ± 30  734 ± 46  2 BCY6136, 176 ± 7  200 ± 8  210 ± 14  224 ± 27  238 ± 21  184 ± 18  244 ± 23  1 mpk, qw 3 BCY6136, 176 ± 18  197 ± 25  168 ± 25  170 ± 26  165 ± 34  96 ± 27 133 ± 35  2 mpk, qw 4 BCY6136, 177 ± 8  197 ± 9  169 ± 7  158 ± 3  148 ± 8  95 ± 16 141 ± 12  3 mpk, qw Days after the start of treatment Gr. Treatment 16 18 21 23 25 28 30 1 Vehicle, qw 821 ± 55  918 ± 91  1024 ± 83  1151 ± 68  1305 ± 57  1407 ± 64  1465 ± 90  2 BCY6136, 276 ± 35  308 ± 44  343 ± 37  390 ± 43  406 ± 48  422 ± 42  425 ± 47  1 mpk, qw 3 BCY6136, 150 ± 52  160 ± 49  190 ± 63  203 ± 65  218 ± 66  201 ± 53  210 ± 60  2 mpk, qw 4 BCY6136, 145 ± 24  164 ± 28  202 ± 28  205 ± 30  201 ± 16  196 ± 21  201 ± 22  3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the NCI-N87 xenograft was calculated based on tumor volume measurements at day 30 after the start of treatment.

TABLE 54 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Group Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 1465 ± 90 — — — 2 BCY6136,  425 ± 47 29.0 80.7 p < 0.001 1 mpk, qw 3 BCY6136,  210 ± 60 14.3 97.4 p < 0.001 2 mpk, qw 4 BCY6136,  201 ± 22 13.7 98.1 p < 0.001 3 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor growth inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the NCI-N87 model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in the FIG. 29 and Tables 53 and 54.

The mean tumor size of vehicle treated mice reached 1465 mm³ on day 30. BCY6136 at 1 mg/kg, qw (TV=425 mm³, TGI=80.7%, p<0.001) and 2 mg/kg, qw (TV=210 mm³, TGI=97.4%, p<0.001) produced significant antitumor activity in a dose-dependent manner, BCY6136 at 3 mg/kg, qw (TV=201 mm³, TGI=98.1%, p<0.001) showed comparable antitumor activity with BCY6136 at 2 mpk.

In this study, no obvious body weight loss was found in all the groups during the treatment schedule.

Study 20: In Vivo Efficacy Study of BCY6136 in Treatment of SK-OV-3 Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of SK-OV-3 xenograft in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule 1 Vehicle 3 — 10 iv Qw 2 ADC 3 3 10 iv Qw 3 BCY6136 3 1 10 iv Qw 4 BCY6136 3 2 10 iv Qw 5 BCY6136 3 3 10 iv Qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The SK-OV-3 tumor cells were maintained in McCoy's 5a medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with SK-OV-3 tumor cells (10×10⁶) with matrigel (1:1) in 0.2 ml of PBS for tumor development. The animals were randomized and treatment was started when the average tumor volume reached approximately 186 mm³. The test article administration and the animal number in each group are shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Test Conc. article Purity (mg/ml) Formulation Vehicle — — 50 mM Acetate 10% sucrose pH5 BCY6136 98.5% 1 Dissolve 3.65 mg BCY6136 in 3.60 ml 50 mM Acetate buffer¹ 0.1 Dilute 90 μl 1 mg/ml BCY6136 stock with 810 μl Acetate buffer¹ 0.2 Dilute 180 μl 1 mg/ml BCY6136 stock with 720 μl Acetate buffer¹ 0.3 Dilute 270 μl 1 mg/ml BCY6136 stock with 630 μl Acetate buffer¹ ADC ADC 0.3 Dilute 69 μl 10.47 mg/ml ADC stock with 2331 μl ADC buffer² ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5 ²ADC buffer: 25 mM Histidine 10% sucrose pH 5.5

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve is shown in FIG. 30 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing SK-OV-3 xenograft is shown in Table 55.

TABLE 55 Tumor volume trace over time Days after the start of treatment Gr. Treatment 0 2 5 7 9 12 14 1 Vehicle, qw 187 ± 16  243 ± 24  313 ± 28  399 ± 37  470 ± 23  606 ± 61  742 ± 103 2 ADC, 187 ± 16  181 ± 15  212 ± 16  263 ± 35  268 ± 14  335 ± 23  353 ± 18  3 mpk, qw 3 BCY6136, 186 ± 23  222 ± 19  293 ± 34  331 ± 21  356 ± 23  440 ± 8  503 ± 28  2 mpk, qw 4 BCY6136, 186 ± 23  170 ± 18  164 ± 28  188 ± 33  180 ± 34  202 ± 29  200 ± 29  2 mpk, qw 5 BCY6136, 184 ± 24  168 ± 18  150 ± 12  164 ± 12  158 ± 8  180 ± 8  187 ± 4  3 mpk, qw Days after the start of treatment Gr. Treatment 16 19 21 23 26 28 1 Vehicle, qw 891 ± 133 1076 ± 185  1173 ± 214  1340 ± 236  1490 ± 273  1560 ± 305  2 ADC, 392 ± 63  449 ± 4  481 ± 27  573 ± 33  647 ± 26  684 ± 111 3 mpk, qw 3 BCY6136, 587 ± 33  702 ± 43  752 ± 26  893 ± 34  1002 ± 68  1035 ± 67  2 mpk, qw 4 BCY6136, 230 ± 46  229 ± 48  231 ± 58  236 ± 49  240 ± 48  277 ± 58  2 mpk, qw 5 BCY6136, 212 ± 17  208 ± 29  204 ± 12  205 ± 17  227 ± 31  254 ± 48  3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the SK-OV-3 xenograft was calculated based on tumor volume measurements at day 28 after the start of treatment.

TABLE 56 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Group Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 1560 ± 305 — — — 2 ADC,  684 ± 111 43.9 63.8 p < 0.01 3 mpk, qw 3 BCY6136, 1035 ± 67 66.4 38.1 p > 0.05 1 mpk, qw 4 BCY6136,  277 ± 58 17.8 93.3 p < 0.001 2 mpk, qw 5 BCY6136,  254 ± 48 16.3 95.0 p < 0.001 3 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor growth inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the SK-OV-3 model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in the FIG. 30 and Tables 55 and 56.

The mean tumor size of vehicle treated mice reached 1560 mm³ on day 28. ADC at 3 mg/kg, qw (TV=684 mm³, TGI=63.8%, p<0.01) showed moderate anti-tumor efficacy. BCY6136 at 1 mg/kg, qw (TV=1035 mm³, TGI=38.1%, p>0.05) didn't show obvious anti-tumor activity. BCY6136 at 2 mg/kg, qw (TV=277 mm³, TGI=93.3%, p<0.001) and 3 mg/kg, qw (TV=254 mm³, TGI=95.0%, p<0.001) produced significant anti-tumor activity.

In this study, no obvious body weight loss was found in all the groups during the treatment schedule.

Study 21: In Vivo Efficacy Study of BCY6136 in Treatment of OE21 Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of OE21 xenograft in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule 1 Vehicle 3 — 10 iv qw 2 BCY6136 3 1 10 iv qw 3 BCY6136 3 2 10 iv qw 4 BCY6136 3 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The OE21 tumor cells were maintained in RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with OE21 tumor cells (5×10⁶) with matrigel (1:1) in 0.2 ml of PBS for tumor development. The animals were randomized and treatment was started when the average tumor volume reached approximately 157 mm³. The test article administration and the animal number in each group are shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10% sucrose pH 5 BCY6136 1 Dissolve 4.295 mg BCY6136 in 4.214 ml Acetate buffer¹ 0.1 Dilute 90 μl 1 mg/ml BCY6136 stock with 810 μl Acetate buffer 0.2 Dilute 180 μl 1 mg/ml BCY6136 stock with 720 μl Acetate buffer 0.3 Dilute 270 μl 1 mg/ml BCY6136 stock with 630 μl Acetate buffer ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve is shown in FIG. 31 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing OE21 xenograft is shown in Table 57.

TABLE 57 Tumor volume trace over time Days after the start of treatment Gr. Treatment 0 2 4 7 9 1 Vehicle, qw 155 ± 9  211 ± 16  291 ± 16  379 ± 14  456 ± 32  2 BCY6136, 159 ± 14  202 ± 28  251 ± 29  282 ± 6  331 ± 19  1 mpk, qw 3 BCY6136, 157 ± 19  197 ± 13  219 ± 6  235 ± 27  268 ± 35  2 mpk, qw 4 BCY6136, 155 ± 19  200 ± 16  197 ± 7  209 ± 11  229 ± 26  3 mpk, qw Days after the start of treatment Gr. 11 14 16 18 21 23 1 539 ± 13  828 ± 42  955 ± 40  1035 ± 58  1250 ± 46  1586 ± 57  2 392 ± 35  609 ± 56  694 ± 44  777 ± 68  1083 ± 85  1155 ± 98  3 243 ± 37  346 ± 78  371 ± 98  396 ± 109 515 ± 94  537 ± 122 4 211 ± 14  289 ± 38  318 ± 53  330 ± 40  474 ± 42  489 ± 51 

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the OE21 xenograft was calculated based on tumor volume measurements at day 23 after the start of treatment.

TABLE 58 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI Group Treatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 1586 ± 57 — — — 2 BCY6136, 1155 ± 98 72.8 30.4 p < 0.05 1 mpk, qw 3 BCY6136,  537 ± 122 33.9 73.4 p < 0.001 2 mpk, qw 4 BCY6136,  489 ± 51 30.8 76.7 p < 0.001 3 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor growth inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the OE21 model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in the FIG. 31 and Tables 57 and 58.

The mean tumor size of vehicle treated mice reached 1586 mm³ on day 23. BCY6136 at 1 mg/kg, qw (TV=1155 mm³, TGI=30.4% p<0.05) showed slight anti-tumor activity. BCY6136 at 2 mg/kg, qw (TV=537 mm³, TGI=73.4%, p<0.001) and 3 mg/kg, qw (TV=489 mm³, TGI=76.7%, p<0.001) produced significant anti-tumor activity.

In this study, no obvious body weight loss was found in all the groups during the treatment schedule.

Study 22: In Vivo Efficacy Test of BCY6136 and BCY6082 in Treatment of MOLP-8 Xenograft in CB17-SCID Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 and BCY6082 in treatment of MOLP-8 xenograft in CB17-SCID mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule 1 Vehicle 3 — 10 iv qw 2 BCY6136 3 1 10 iv qw 3 BCY6136 3 2 10 iv qw 4 BCY6136 3 3 10 iv qw 5 BCY6082 3 1 10 iv qw 6 BCY6082 3 2 10 iv qw 7 BCY6082 3 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The MOLP-8 tumor cells were maintained in vitro as a monolayer culture in RMPI-1640 supplemented with 20% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were routinely subcultured by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with MOLP-8 tumor cells (10×10⁶) in 0.2 ml PBS with 50% matrigel for tumor development. 36 animals were randomized when the average tumor volume reached 141 mm³. The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Concentration Treatment (mg/ml) Formulation Vehicle — 50 mM Acetate, 10% sucrose pH = 5 BCY6136 0.1 Dilute 90 μl 1 mg/ml BCY6136 stocks* with 810 μl buffer*** 0.2 Dilute 180 μl 1 mg/ml BCY6136 stocks* with 720 μl buffer*** 0.3 Dilute 270 μl 1 mg/ml BCY6136 stocks* with 630 μl buffer*** BCY6082 0.1 Dilute 90 μl 1 mg/ml BCY6082 stocks** with 810 μl buffer*** 0.2 Dilute 180 μl 1 mg/ml BCY6082 stocks** with 720 μl buffer*** 0.3 Dilute 270 μl 1 mg/ml BCY6082 stocks** with 630 μl buffer*** *BCY6136 stocks: 10.93 mg BCY6136 dissolved to 10.93 mL 50 mM Acetate, 10% sucrose, pH = 5, and separated into individual tubes and stored at −80° C. **BCY6082 stocks: 2.43 mg BCY6136 dissolved to 2.43 mL 50 mM Acetate, 10% sucrose, pH = 5, and separated into individual tubes and stored at −80° C. ***Buffer: 50 mM Acetate, 10% sucrose pH = 5

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIGS. 32 and 33 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female CB17-SCID mice bearing MOLP-8 xenograft is shown in Table 59.

TABLE 59 Tumor volume trace over time Days after the start of treatment Gr. Treatment 0 2 4 7 9 11 14 1 Vehicle, qw 139 ± 2  375 ± 36  604 ± 28  984 ± 88  1451 ± 135  1981 ± 196  2528 ± 295  2 BCY6136, 143 ± 13  299 ± 6  444 ± 49  576 ± 31  806 ± 85  1132 ± 170  1446 ± 234  1 mpk, qw 3 BCY6136, 140 ± 15  271 ± 43  250 ± 2  509 ± 23  662 ± 78  873 ± 49  1218 ± 144  2 mpk, qw 4 BCY6136, 142 ± 19  239 ± 67  197 ± 20  342 ± 78  425 ± 90  693 ± 133 938 ± 155 3 mpk, qw 5 BCY6082, 142 ± 4  303 ± 49  456 ± 83  809 ± 169 1365 ± 277  1708 ± 190  2296 ± 511  1 mpk, qw 6 BCY6082, 139 ± 5  273 ± 46  428 ± 18  682 ± 50  945 ± 73  1240 ± 85  1554 ± 84  2 mpk, qw 7 BCY6082, 142 ± 4  219 ± 7  369 ± 77  471 ± 81  656 ± 115 997 ± 212 1321 ± 336  3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 and BCY6082 in the MOLP-8 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

TABLE 60 Tumor growth inhibition analysis Tumor P value Volume T/C^(b) TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw 2528 ± 295 — — — 2 BCY6136, 1446 ± 234 57.2 45.5 p > 0.05 1 mpk, qw 3 BCY6136, 1218 ± 144 48.2 54.9 p < 0.05 2 mpk, qw 4 BCY6136,  938 ± 155 37.1 66.7 p < 0.01 3 mpk, qw 5 BCY6082, 2296 ± 511 90.8  9.8 p > 0.05 1 mpk, qw 6 BCY6082, 1554 ± 84 61.5 40.8 p > 0.05 2 mpk, qw 7 BCY6082, 1321 ± 336 52.3 50.6 p < 0.05 3 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 and BCY6082 in the MOLP-8 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIGS. 32 and 33 and Tables 59 and 60.

The mean tumor size of vehicle treated mice reached 2528 mm³ on day 14. BCY6136 at 1 mg/kg (TV=1146 mm³, TGI=45.5%, p>0.05), 2 mg/kg (TV=1218 mm³, TGI=54.9%, p<0.05) and 3 mg/kg (TV=938 mm³, TGI=66.7%, p<0.01) produced dose-dependent antitumor activity, but all of dosage didn't regress the tumors in MOLP-8 xenografts.

BCY6082 at 1 mg/kg (TV=2296 mm³, TGI=9.8%, p>0.05) and 2 mg/kg (TV=1554 mm³, TGI=40.8%, p>0.05) didn't show obvious anti-tumor activity. BCY6082 at 3 mg/kg inhibited the tumor growth significantly (TV=1321 mm³, TGI=50.6%, p<0.05), but didn't regress the tumors in MOLP-8 xenografts.

In this study, all of mice maintained the bodyweight well.

Study 23: In Vivo Efficacy Test of BCYs in Treatment of HT1080 Xenograft in BALB/c Nude Mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCYs in treatment of HT1080 xenograft model in BALB/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) Route Schedule  1 Vehicle 3 — 10 iv qw  2 BCY6082 3 2 10 iv qw  3 BCY6031 3 2 10 iv qw  4 BCY6173 3 1 10 iv qw  5 BCY6173 3 2 10 iv qw  6 BCY6173 3 3 10 iv qw  7 BCY6135 3 1 10 iv qw  8 BCY6135 3 2 10 iv qw  9 BCY6135 3 3 10 iv qw 10 BCY6033 3 3 10 iv qw 11 BCY6033 3 5 10 iv qw 12 BCY6136 3 2 10 iv qw 13 BCY6136 3 3 10 iv qw 14 BCY6136 3 5 10 iv qw 15 BCY6174 3 1 10 iv qw 16 BCY6174 3 2 10 iv qw 17 BCY6174 3 3 10 iv qw 18 BCY6175 3 1 10 iv qw 19 BCY6175 3 2 10 iv qw 20 BCY6175 3 3 10 iv qw 21 ADC 3 3 10 iv qw Note: n: animal number; Dosing volume: adjust dosing volume based on body weight 10 μl/g.

(c) Experimental Methods and Procedures

(i) Cell Culture

The HT1608 tumor cells will be maintained in medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells will be routinely subcultured twice weekly. The cells growing in an exponential growth phase will be harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse will be inoculated subcutaneously at the right flank with HT108 tumor cells (5*10⁶) for tumor development. The animals will be randomized and treatment will be started when the average tumor volume reaches approximately 150-200 mm³. The test article administration and the animal numbers in each group are shown in the following experimental design table.

(iii) Testing Article Formulation Preparation

Dose Treatment (mg/ml) Formulation Vehicle — 50 mM Acetate/acetic acid pH 5 10% sucrose BCY6082 0.2 Dilute 160 μl 1 mg/ml BCY6082 stock with 640 μl buffer BCY6031 0.2 Dilute 180 μl 1 mg/ml BCY6031 stock with 720 μl buffer BCY6173 1 Dissolve 2.13 mg BCY6173 with 2.04 ml buffer 0.1 Dilute 90 μl 1 mg/ml BCY6173 stock with 810 μl buffer 0.2 Dilute 180 μl 1 mg/ml BCY6173 stock with 720 μl buffer 0.3 Dilute 270 μl 1 mg/ml BCY6173 stock with 630 μl buffer BCY6135 1 Dissolve 2 mg BCY6135 with 1.9 ml buffer 0.1 Dilute 90 μl 1 mg/ml BCY6135 stock with 810 μl buffer 0.2 Dilute 180 μl 1 mg/ml BCY6135 stock with 720 μl buffer 0.3 Dilute 270 μl 1 mg/ml BCY6135 stock with 630 μl buffer BCY6033 0.3 Dilute 270 μl 1 mg/ml BCY6033 stock with 630 μl buffer 0.5 Dilute 450 μl 1 mg/ml BCY6033 stock with 450 μl buffer BCY6136 0.2 Dilute 200 μl 1 mg/ml BCY6136 stock with 800 μl buffer 0.3 Dilute 300 μl 1 mg/ml BCY6136 stock with 700 μl buffer 0.5 Dilute 500 μl 1 mg/ml BCY6136 stock with 500 μl buffer BCY6174 1 Dissolve 2.69 mg BCY6174 with 2.677 ml buffer 0.1 Dilute 90 μl 1 mg/ml BCY6174 stock with 810 μl buffer 0.2 Dilute 180 μl 1 mg/ml BCY6174 stock with 720 μl buffer 0.3 Dilute 270 μl 1 mg/ml BCY6174 stock with 630 μl buffer BCY6175 1 Dissolve 2 mg BCY6175 with 1.924 ml buffer 0.1 Dilute 90 μl 1 mg/ml BCY6175 stock with 810 μl buffer 0.2 Dilute 180 μl 1 mg/ml BCY6175 stock with 720 μl buffer 0.3 Dilute 270 μl 1 mg/ml BCY6175 stock with 630 μl buffer ADC 0.3 Dilute 25.78 μl 10.47 mg/ml ADC stock with 874.22 μl 25 mM Histidine pH 7 10% sucrose

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIGS. 34 to 42 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing HT1080 xenograft is shown in Table 61.

TABLE 61 Tumor volume trace over time Days after the start of treatment Gr Treatment 0 2 4 7 9 11 14 1 Vehicle, qw 179 ± 22 312 ± 84  529 ± 135 886 ± 207 1185 ± 172  1467 ± 224  1737 ± 258  2 BCY6082, 177 ± 16 183 ± 31  99 ± 27 61 ± 17 33 ± 10 12 ± 5  5 ± 3 2 mpk, qw 3 BCY6031, 177 ± 24 215 ± 35  133 ± 37  63 ± 31 53 ± 37 45 ± 36 71 ± 67 2 mpk, qw 4 BCY6173 178 ± 26 276 ± 8  328 ± 73  594 ± 62  745 ± 22  960 ± 53  1074 ± 150  1 mpk, qw 5 BCY6173, 178 ± 28 277 ± 61  262 ± 125 309 ± 238 425 ± 334 436 ± 323 480 ± 347 2 mpk, qw 6 BCY6173, 179 ± 43 182 ± 71  133 ± 88  87 ± 68 77 ± 65 60 ± 54 47 ± 42 3 mpk, qw 7 BCY6135 178 ± 22 267 ± 66  262 ± 58  436 ± 67  599 ± 89  703 ± 36  871 ± 28  1 mpk, qw 8 BCY6135 178 ± 23 176 ± 48  117 ± 43  70 ± 23 67 ± 23 52 ± 21 62 ± 7  2 mpk, qw 9 BCY6135 177 ± 39 178 ± 79  92 ± 67 62 ± 46 62 ± 51 57 ± 51 44 ± 40 3 mpk, qw 10 BCY6033 178 ± 26 186 ± 34  79 ± 30 29 ± 15 12 ± 8  6 ± 4 9 ± 7 3 mpk, qw 11 BCY6033 178 ± 36 117 ± 20  41 ± 10 12 ± 4  6 ± 2 4 ± 0 0 ± 0 5 mpk, qw 12 BCY6136 178 ± 19 249 ± 22  115 ± 8  126 ± 53  158 ± 71  140 ± 89  245 ± 116 2mpk, qw 13 BCY6136 178 ± 36 168 ± 21  72 ± 18 22 ± 7  21 ± 15 8 ± 6 3 ± 2 3 mpk, qw 14 BCY6136 178 ± 26 165 ± 33  52 ± 10 18 ± 7  9 ± 4 5 ± 2 2 ± 1 5 mpk, qw 15 BCY6174 180 ± 35 231 ± 19  226 ± 29  432 ± 37  602 ± 63  742 ± 62  1066 ± 130  1 mpk, qw 16 BCY6174 178 ± 31 203 ± 50  123 ± 29  216 ± 47  291 ± 40  326 ± 68  532 ± 91  2 mpk, qw 17 BCY6174 178 ± 33 195 ± 13  110 ± 39  58 ± 23 34 ± 17 21 ± 11 11 ± 7  3 mpk, qw 18 BCY6175 178 ± 27 248 ± 62  244 ± 74  347 ± 18  435 ± 18  558 ± 38  769 ± 26  1 mpk, qw 19 BCY6175 178 ± 22 223 ± 42v 158 ± 59  116 ± 35  156 ± 52  166 ± 51  295 ± 88  2 mpk, qw 20 BCY6175 179 ± 39 189 ± 48  116 ± 50  43 ± 18 33 ± 18 25 ± 13 11 ± 9  3 mpk, qw 21 ADC 180 ± 26 158 ± 30  58 ± 8  18 ± 2  7 ± 1 2 ± 2 0 ± 0 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCYs in the HT1080 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

TABLE 62 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI P value Gr Treatment (mm³)^(a) (%) (%) compare 1 Vehicle, qw 1737 ± 258 — — — 2 BCY6082,   5 ± 3  0.3 111.1 p < 0.01 2 mpk, 3 BCY6031,  71 ± 67  4.1 106.8 p < 0.01 2 mpk, 4 BCY6173, 1074 ± 150 61.8  42.5 p > 0.05 1 mpk, 5 BCY6173,  480 ± 347 27.6  80.6 p < 0.05 2 mpk, 6 BCY6173,  47 ± 42  2.7 108.4 p < 0.01 3 mpk, 7 BCY6135,  871 ± 28 50.1  55.5 p < 0.01 1 mpk, 8 BCY6135,  62 ± 7  3.5 107.5 p < 0.001 2 mpk, 9 BCY6135,  44 ± 40  2.5 108.6 p < 0.001 3 mpk, 10 BCY6033,   9 ± 7  0.5 110.8 p < 0.001 3 mpk, 11 BCY6033,   0 ± 0  0.0 111.4 p < 0.001 5 mpk, 12 BCY6136,  245 ± 116 14.1  95.7 p < 0.001 2 mpk, qw 13 BCY6136,   3 ± 2  0.2 111.2 p < 0.001 3 mpk, 14 BCY6136,   2 ± 1  0.1 111.3 p < 0.001 5 mpk, 15 BCY6174, 1066 ± 130 61.4  43.1 p < 0.05 1 mpk, 16 BCY6174,  532 ± 91 30.6  77.3 p < 0.01 2 mpk, qw 17 BCY6174,  11 ± 7  0.6 110.7 p < 0.001 3 mpk, 18 BCY6175,  769 ± 26 44.3  62.1 p < 0.01 1 mpk, 19 BCY6175,  295 ± 88 17.0  92.5 p < 0.001 2 mpk, 20 BCY6175,  11 ± 9  0.6 110.8 p < 0.001 3 mpk, 21 ADC,   0 ± 0  0.0 111.5 — 3 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCYs in the HT1080 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the FIGS. 34 to 42 and Tables 61 and 62.

The mean tumor size of vehicle treated mice reached 1737 mm³ on day 14.

BCY6082 at 2 mg/kg, qw (TV=5 mm³, TGI=111.1%, p<0.01) and BCY6031 at 2 mg/kg qw (TV=7 mm³, TGI=106.8%, p<0.01) showed potent anti-tumor activity.

BCY6173 at 1 mg/kg, qw (TV=1074 mm³, TGI=42.5%, p>0.05), 2 mg/kg, qw (TV=480 mm³, TGI=80.6%, p<0.05) and 3 mg/kg, qw (TV=7 mm³, TGI=108.4%, p<0.01) produced dose-dependent antitumor activity.

BCY6135 at 1 mg/kg, qw (TV=871 mm³, TGI=55.5%, p<0.01), 2 mg/kg, qw (TV=62 mm³, TGI=107.5%, p<0.001) and 3 mg/kg, qw (TV=44 mm³, TGI=108.6%, p<0.001) produced dose-dependent antitumor activity.

BCY6033 at 3 mg/kg, qw (TV=9 mm³, TGI=110.8%, p<0.001) and 5 mg/kg, qw (TV=0 mm³, TGI=111.4%, p<0.001) showed potent anti-tumor activity, and completely eradicated the tumors by day 14 at 5 mg/kg.

BCY6136 at 2 mg/kg, qw (TV=345 mm³, TGI=95.7%, p<0.001), 3 mg/kg, qw (TV=3 mm³, TGI=111.2%, p<0.001) and 5 mg/kg, qw (TV=2 mm³, TGI=111.3%, p<0.001) showed potent anti-tumor activity.

BCY6174 at 1 mg/kg, qw (TV=1066 mm³, TGI=43.1%, p<0.05), 2 mg/kg, qw (TV=532 mm³, TGI=77.3%, p<0.01) and 3 mg/kg, qw (TV=11 mm³, TGI=110.7%, p<0.001) produced dose-dependent antitumor activity.

BCY6175 at 1 mg/kg, qw (TV=769 mm³, TGI=62.1%, p<0.01), 2 mg/kg, qw (TV=295 mm³, TGI=92.5%, p<0.001) and 3 mg/kg, qw (TV=11 mm³, TGI=110.8%, p<0.001) produced dose-dependent antitumor activity.

ADC at 3 mg/kg, qw (TV=0 mm³, TGI=111.5%) completely eradicated the tumors by day 14.

Study 24: Investigation of Association Between Copy Number Variation (CNV) and Gene Expression for EphA2 from Multiple Tumour Types

Methods

-   -   1. Select all studies in cBioPortal (http://www.cbioportal.org/)         and search for EPHA2.         -   (a) Remove provisional studies.         -   (b) Deselect studies with overlapping samples to prevent             sample bias (based on warning in cBioPortal)—always keep             PanCancer study if this is an option.         -   (c) Studies selected for analysis (Table 63).

TABLE 63 Studies analysed from cBioPortal and units in study Study Name Units Breast Invasive Carcinoma (TCGA, mRNA Expression Batch Normalized/Merged PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Lung Squamous Cell Carcinoma mRNA Expression Batch Normalized/Merged (TCGA, PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Kidney Renal Papillary Cell Carcinoma mRNA Expression, RSEM (Batch normalized (TCGA, PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Kidney Renal Clear Cell Carcinoma mRNA Expression, RSEM (Batch normalized (TCGA, PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Colon Adenocarcinoma (TCGA, RSEM (Batch normalized from IIlumina PanCancer Atlas) HiSeq_RNASeqV2) Head and Neck Squamous Cell mRNA Expression, RSEM (Batch normalized Carcinoma (TCGA, PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Bladder Urothelial Carcinoma (TCGA, RSEM (Batch normalized from IIlumina PanCancer Atlas) HiSeq_RNASeqV2) Uveal Melanoma (TCGA, PanCancer mRNA Expression Batch Normalized/Merged Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Lung Adenocarcinoma (TCGA, mRNA Expression, RSEM (Batch normalized PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Ovarian Serous Cystadenocarcinoma mRNA Expression Batch Normalized/Merged (TCGA, PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Breast Cancer (METABRIC, Nature mRNA expression (microarray) 2012 & Nat Commun 2016) Mesothelioma (TCGA, PanCancer mRNA Expression Batch Normalized/Merged Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Colorectal Adenocarcinoma (TCGA, RNA Seq RPKM Nature 2012) Cervical Squamous Cell Carcinoma RSEM (Batch normalized from IIlumina (TCGA, PanCancer Atlas) HiSeq_RNASeqV2) Sarcoma (TCGA, PanCancer Atlas) mRNA Expression Batch Normalized/Merged from IIlumina HiSeq_RNASeqV2 syn4976369 Cancer Cell Line Encyclopedia mRNA expression (microarray) (Novartis/Broad, Nature 2012) Rectum Adenocarcinoma (TCGA, mRNA Expression Batch Normalized/Merged PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Liver Hepatocellular Carcinoma (TCGA, EPHA2: mRNA Expression, RSEM (Batch PanCancer Atlas) normalized from IIlumina HiSeq_RNASeqV2) Stomach Adenocarcinoma (TCGA, mRNA Expression Batch Normalized/Merged PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Uterine Corpus Endometrial Carcinoma mRNA Expression Batch Normalized/Merged (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2 syn4976369 Skin Cutaneous Melanoma (TCGA, mRNA Expression Batch Normalized/Merged PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Prostate Adenocarcinoma (TCGA, mRNA Expression, RSEM (Batch normalized PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Kidney Chromophobe (TCGA, mRNA Expression, RSEM (Batch normalized PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Pediatric Wilms' Tumor (TARGET, Epha2: mRNA expression (RNA-Seq RPKM) 2018) Pheochromocytoma and mRNA Expression Batch Normalized/Merged Paraganglioma (TCGA, PanCancer from IIlumina HiSeq_RNASeqV2 syn4976369 Atlas) Thyroid Carcinoma (TCGA, PanCancer mRNA Expression Batch Normalized/Merged Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Esophageal Adenocarcinoma (TCGA, RSEM (Batch normalized from IIlumina PanCancer Atlas) HiSeq_RNASeqV2) Cholangiocarcinoma (TCGA, RSEM (Batch normalized from IIlumina PanCancer Atlas) HiSeq_RNASeqV2) Brain Lower Grade Glioma (TCGA, RSEM (Batch normalized from IIlumina PanCancer Atlas) HiSeq_RNASeqV2) Thymoma (TCGA, PanCancer Atlas) mRNA Expression Batch Normalized/Merged from IIlumina HiSeq_RNASeqV2 syn4976369 Pediatric Acute Lymphoid Leukemia- Epha2: mRNA expression (RNA-Seq RPKM) Phase II (TARGET, 2018) Diffuse Large B-Cell Lymphoma mRNA Expression, RSEM (Batch normalized (TCGA, PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Glioblastoma Multiforme (TCGA, mRNA Expression, RSEM (Batch normalized PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Metastatic Prostate Cancer, SU2C/PCF mRNA expression/capture (RNA Seq RPKM) Dream Team (Robinson et al., Cell 2015) Acute Myeloid Leukemia (TCGA, mRNA Expression, RSEM (Batch normalized PanCancer Atlas) from IIlumina HiSeq_RNASeqV2) Testicular Germ Cell Tumors (TCGA, mRNA Expression Batch Normalized/Merged PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Adrenocortical Carcinoma (TCGA, RSEM (Batch normalized from IIlumina PanCancer Atlas) HiSeq_RNASeqV2) Uterine Carcinosarcoma (TCGA, mRNA Expression Batch Normalized/Merged PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Pancreatic Adenocarcinoma (TCGA, mRNA Expression Batch Normalized/Merged PanCancer Atlas) from IIlumina HiSeq_RNASeqV2 syn4976369 Prostate Adenocarcinoma (MSKCC, mRNA Expression Cancer Cell 2010) Prostate Adenocarcinoma (Fred mRNA expression Hutchinson CRC, Nat Med 2016)

-   -   2. Export CNV and RNA expression data from cBioPortal.     -   3. Test if CNVs are statistically significantly associated with         changes in mRNA expression for EphA2 (log 2 not applied).         -   (a) Run non-parametric Kruskal-Wallis test in GraphPad Prism             (7.04) and R/R studio (threshold for significance: p<0.01).             -   (i) GraphPad Prism: set up column table, run                 non-parametric test with no matching or pairing and do                 not assume Gaussian distribution.             -   (ii) Packages used in R:                 -   1. XLConnect                 -   2. dplyr                 -   3. Kruskal-Wallis Rank Sum Test: Kruskal.test.     -   4. Adjust for multiple comparisons (include all possible         comparisons even if n=1 within a group) in R/Rstudio using         Dunn's test (threshold for significance: p<0.025).         -   (a) dunn.test with multiple comparison method=“bonferonni”.

Results

The results are shown in Table 64 below. Across 41 publicly available datasets compiled in cBioPortal that report both Copy Number Variation (CNV) and mRNA gene expression for EphA2, there are numerous cancer types where cases have been reported with EphA2 shallow-deletions (<2 copies). Although less common, in these same cancer types a subset of tumors harbored EphA2 deep deletions (>1 copy loss or biallelic loss), EphA2 gains (2-3 copies) or EphA2 amplifications (>3 copies). Indications where >33% of tumors had either shallow-deletions or deep deletions in EphA2 included: kidney chromophobe, cholangiocarcinoma, pheochromocytoma and paraganglioma, lung squamous cancer, breast, rectum, brain lower grade glioma, liver, adrenocortical carcinoma, mesothelioma, esophageal adenocarcinoma and colon cancer. In contrast, there were no studies where >33% of samples had either gains or amplification in EphA2. Taken together these results demonstrate that deletions in EphA2 DNA are found across a variety of indications.

Approximately one third of all samples analyzed in the 41 studies harbored EphA2 CNVs. Based on this high percentage of CNVs across studies, and the high percentage of shallow deletions within specific tumor types, statistical testing was performed to identify possible associations between copy number changes and RNA expression. Tumors per indication were allocated to 1 of 5 classes:

-   -   a) Deep deletion;     -   b) Shallow deletion;     -   c) Diploid;     -   d) Gain; or     -   e) Amplification.

Kruskall-Wallis testing was then performed to detect if the distributions of mRNA expression values per classes differed between classes (P<0.01). For those TCGA data sets with P<0.01 and to identify which classes were different to one another post-hoc testing was performed by calculating Z-statistics with adjusted P-values calculated (Bonferroni). For simplicity of interpretation pair-wise comparisons vs. diploid per indication were reviewed (although all pair-wise P-values were calculated). 19/41 of these studies had a Kruskall-Wallis p-value of <0.01 demonstrating that copy number is statistically significantly associated with RNA expression. Of these 19 studies, 17 of them had a Bonferroni adjusted P<0.025 for Diploid vs. Shallow Deletion indicating an association of decreased EphA2 mRNA expression with decreased EphA2 copy number. Only 2 of these 19 studies had a Bonferroni adjusted P<0.025 for Diploid vs. Gain and both were breast cancer studies. Furthermore, one of these breast cancer studies (Breast Invasive Carcinoma (TCGA, PanCancer Atlas)) had a Bonferroni adjusted P<0.025 for both Diploid vs. Shallow Deletion and Diploid vs. Gain suggesting that copy number alterations may have a strong impact on EphA2 RNA expression in breast cancer.

The central dogma of genetics suggests that reduced copy number in EphA2 lead to reduced RNA and protein expression. Therefore, the observed associations between copy number loss of EphA2 and reduced mRNA expression in a variety of tumor types suggest that EphA2 protein expression may also be reduced. Similarly, copy number gains of EphA2 in breast cancer that were associated with increased mRNA expression may also suggest increased EphA2 protein expression. Moreover, higher EphA2 protein expression (measured by FACS) is associated with increased efficacy of certain EphA2 bicyclic drug conjugates of the invention (measured by tumor volume) in preclinical in vivo models. Taken together if copy number alterations that are associated with mRNA expression changes do predict protein expression levels then patients with tumors containing copy number deletions of EphA2 may be less likely to respond to EphA2 bicyclic drug conjugates of the invention. Similarly, if patients with tumor copy number gains in EphA2 (e.g. breast cancer) it is possible that these patients would be more likely to respond to EphA2 bicyclic drug conjugates of the invention. Therefore, if patients were stratified by EphA2 copy number status, then this information could be used to both exclude and select patients for treatment with EphA2 bicyclic drug conjugates of the invention to increase efficacy.

TABLE 64 Results of Investigation of Association between Copy Number Variation (CNV) and gene expression for EphA2 Number of samples/group (n = X) Deep Shallow Study name Units deletion deletion Diploid Gain Amplification Breast Invasive mRNA 5 415 511 61 2 Carcinoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Lung Squamous mRNA 3 207 201 55 0 Cell Carcinoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Kidney Renal mRNA 1 48 224 0 1 Papillary Cell Expression, Carcinoma RSEM (Batch (TCGA, normalized PanCancer from IIlumina Atlas) HiSeq_RNAS eqV2) Kidney Renal mRNA 0 69 278 5 0 Clear Cell Expression, Carcinoma RSEM (Batch (TCGA, normalized PanCancer from IIlumina Atlas) HiSeq_RNAS eqV2) Colon RSEM (Batch 3 132 245 8 0 Adenocarcinoma normalized (TCGA, from IIlumina PanCancer HiSeq_RNAS Atlas) eqV2) Head and Neck mRNA 3 86 345 54 0 Squamous Cell Expression, Carcinoma RSEM (Batch (TCGA, normalized PanCancer from IIlumina Atlas) HiSeq_RNAS eqV2) Bladder RSEM (Batch 0 73 245 80 4 Urothelial normalized Carcinoma from IIlumina (TCGA, HiSeq_RNAS PanCancer eqV2) Atlas) Uveal mRNA 0 24 56 0 0 Melanoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Lung mRNA 1 115 263 121 3 Adenocarcinoma Expression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IIlumina HiSeq_RNAS eqV2) Ovarian Serous mRNA 0 59 78 60 4 Cystadenocarci Expression noma (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Breast Cancer mRNA 1 491 1349 25 0 (METABRIC, expression Nature 2012 & (microarray) Nat Commun 2016) Mesothelioma mRNA 0 29 50 3 0 (TCGA, Expression PanCancer Batch Atlas) Normalized/ Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Colorectal RNA Seq 0 53 138 2 0 Adenocarcinoma RPKM (TCGA, Nature 2012) Cervical RSEM (Batch 1 31 167 76 0 Squamous Cell normalized Carcinoma from IIlumina (TCGA, HiSeq_RNAS PanCancer eqV2) Atlas) Sarcoma mRNA 0 43 113 70 4 (TCGA, Expression PanCancer Batch Atlas) Normalized/ Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Cancer Cell Line mRNA 17 279 418 150 13 Encyclopedia expression (Novartis/Broad, (microarray) Nature 2012) Rectum mRNA 1 54 78 3 0 Adenocarcinoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Liver EPHA2: 1 130 194 21 2 Hepatocellular mRNA Carcinoma Expression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IIlumina HiSeq_RNAS eqV2) Stomach mRNA 2 90 264 44 7 Adenocarcinoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Uterine Corpus mRNA 3 61 395 43 5 Endometrial Expression Carcinoma Batch (TCGA, Normalized/ PanCancer Merged from Atlas) IIlumina HiSeq_RNAS eqV2 syn4976369 Skin Cutaneous mRNA 2 70 216 72 3 Melanoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Prostate mRNA 0 44 438 4 1 Adenocarcinoma Expression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IIlumina HiSeq_RNAS eqV2) Kidney mRNA 0 52 12 1 0 Chromophobe Expression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IIlumina HiSeq_RNAS eqV2) Pediatric Wilms’ Epha2: 0 22 74 5 0 Tumor mRNA (TARGET, expression 2018) (RNA-Seq RPKM) Pheochromocytoma mRNA 4 96 60 1 0 Paraganglioma and Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Thyroid mRNA 0 4 474 2 0 Carcinoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Esophageal RSEM (Batch 1 64 83 32 1 Adenocarcinoma normalized (TCGA, from IIlumina PanCancer HiSeq_RNAS Atlas) eqV2) Cholangiocarcin RSEM (Batch 2 27 7 0 0 oma (TCGA, normalized PanCancer from IIlumina Atlas) HiSeq_RNAS eqV2) Brain Lower RSEM (Batch 0 191 303 13 0 Grade Glioma normalized (TCGA, from Illumina PanCancer HiSeq_RNAS Atlas) eqV2) Thymoma mRNA 0 8 110 1 0 (TCGA, Expression PanCancer Batch Atlas) Normalized/ Merged from Illumina HiSeq_RNAS eqV2 syn4976369 Pediatric Acute Epha2: 1 6 70 4 0 Lymphoid mRNA Leukemia- expression Phase II (RNA-Seq (TARGET, RPKM) 2018) Diffuse Large B- mRNA 0 4 33 0 0 Cell Lymphoma Expression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IIlumina HiSeq_RNAS eqV2) Glioblastoma mRNA 0 13 104 28 0 Multiforme Expression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IIlumina HiSeq_RNAS eqV2) Metastatic mRNA 2 21 87 7 0 Prostate expression/ Cancer, capture (RNA SU2C/PCF Seq RPKM) Dream Team (Robinson et al., Cell 2015) Acute Myeloid mRNA 0 1 160 4 0 Leukemia Expression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IIlumina HiSeq_RNAS eqV2) Testicular Germ mRNA 1 29 92 22 0 Cell Tumors Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Adrenocortical RSEM (Batch 0 28 47 1 0 Carcinoma normalized (TCGA, from IIlumina PanCancer HiSeq_RNAS Atlas) eqV2) Uterine mRNA 0 16 22 16 2 Carcinosarcoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Pancreatic mRNA 2 50 106 9 1 Adenocarcinoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IIlumina HiSeq_RNAS eqV2 syn4976369 Prostate mRNA 0 5 77 3 0 Adenocarcinoma Expression (MSKCC, Cancer Cell 2010) Prostate mRNA 0 39 84 10 0 Adenocarcinoma expression (Fred Hutchinson CRC, Nat Med 2016) Pairwise comparison, Z statistic Kruskal-wallis test (adjusted p-value), Bonferonni Kruskal- Deep Diploid- Ampli- wallis Deletion- Shallow Diploid- fication- Study name statstic p-value Diploid deletion Gain Diploid Breast Invasive 80.816 <2.2e−16 0.176118 6.460580 −4.603180 0.713978 Carcinoma (1.0000) (0.0000)* (0.0000)* (1.0000) (TCGA, PanCancer Atlas) Lung Squamous 52.942 1.89E−11 −1.584610 6.786501 −0.019607 N/A Cell Carcinoma (0.3392) (0.0000)* (1.0000) (TCGA, PanCancer Atlas) Kidney Renal 42.161 3.71E−09 −1.586207 6.097375 N/A 1.549107 Papillary Cell (0.3381)* (0.0000)* (0.3641) Carcinoma (TCGA, PanCancer Atlas) Kidney Renal 38.342 4.72E−09 N/A 6.133219 −0.487059 N/A Clear Cell (0.0000)* (0.9393) Carcinoma (TCGA, PanCancer Atlas) Colon 35.397 1.00E−07 −2.158194 5.670600 0.781046 N/A Adenocarcinoma (0.0927) (0.0000)* (1.0000) (TCGA, PanCancer Atlas) Head and Neck 32.72 3.69E−07 −2.444914 4.680789 −1.530670 N/A Squamous Cell (0.0435) (0.0000)* (0.3776) Carcinoma (TCGA, PanCancer Atlas) Bladder 28.906 2.34E−06 N/A 5.203251 0.211744 0.581704 Urothelial (0.0000)* (1.0000) (1.0000) Carcinoma (TCGA, PanCancer Atlas) Uveal 21.051 4.47E−06 N/A 4.588095 N/A N/A Melanoma (0.0000)* (TCGA, PanCancer Atlas) Lung 28.874 8.29E−06 −0.690460 4.280100 −0.626707 2.276458 Adenocarcinoma (1.0000) (0.0001)* (1.0000) (0.1141) (TCGA, PanCancer Atlas) Ovarian Serous 25.349 1.31E−05 N/A 4.390097 −0.239249 0.240543 Cystadenocarci (0.0000)* (1.0000) (1.0000) noma (TCGA, PanCancer Atlas) Breast Cancer 23.875 2.65E−05 0.568937 2.274564 −4.115288 N/A (METABRIC, (1.0000) (0.0688) (0.0001)* Nature 2012 & Nat Commun 2016) Mesothelioma 18.866 8.00E−05 N/A 4.319425 0.170478 N/A (TCGA, (0.0000)* (1.0000) PanCancer Atlas) Colorectal 18.847 8.08E−05 N/A 4.298092 −0.338975 N/A Adenocarcinoma (0.0000)* (1.0000) (TCGA, Nature 2012) Cervical 19.435 2.22E−04 −1.618248 3.429609 −1.446339 N/A Squamous Cell (0.3168) (0.0018)* (0.4442) Carcinoma (TCGA, PanCancer Atlas) Sarcoma 19.389 2.27E−04 N/A 3.666949 −0.852454 0.953027 (TCGA, (0.0007)* (1.0000) (1.0000) PanCancer Atlas) Cancer Cell Line 20.977 0.00032 −2.084879 −3.615935 −2.007004 −0.108880 Encyclopedia (0.1854) (0.0015)* (0.2237) (1.0000) (Novartis/Broad, Nature 2012) Rectum 18.215 0.0003971 −1.926519 3.877166 1.167400 N/A Adenocarcinoma (0.1621) (0.0003)* (0.7291) (TCGA, PanCancer Atlas) Liver 15.514 0.003745 0.302341 3.697218 −0.336659 0.454454 Hepatocellular (1.0000) (0.0011)* (1.0000) (1.0000) Carcinoma (TCGA, PanCancer Atlas) Stomach 13.966 0.007404 −2.072978 1.606072 −1.750466 1.602806 Adenocarcinoma (0.1909) (0.5413) (0.4002) (0.5449) (TCGA, PanCancer Atlas) Uterine Corpus 12.916 0.0117 −1.905863 1.039307 −1.597383 2.268798 Endometrial (0.2833) (1.0000) (0.5509) (0.1164) Carcinoma (TCGA, PanCancer Atlas) Skin Cutaneous 12.242 0.01564 1.094526 2.674493 0.095966 1.692628 Melanoma (1.0000) (0.0374) (1.0000) (0.4526) (TCGA, PanCancer Atlas) Prostate 10.112 0.01764 N/A 2.905502 1.374609 −0.082790 Adenocarcinoma (0.0110)* (0.5078) (1.0000) (TCGA, PanCancer Atlas) Kidney 7.8781 0.01947 N/A 2.498340 1.863169 N/A Chromophobe (0.0187)* (0.0937) (TCGA, PanCancer Atlas) Pediatric Wilms’ 7.4912 0.02362 N/A 2.690766 −0.173274 N/A Tumor (0.0107)* (1.0000) (TARGET, 2018) Pheochromocytoma 8.8074 0.03196 −1.411567 2.201344 1.946134 N/A Paraganglioma (0.4742) (0.0831) (0.1549) (TCGA, PanCancer Atlas) Thyroid 5.1773 0.08 N/A 2.221884 0.503577 N/A Carcinoma (0.0394) (0.9218) (TCGA, PanCancer Atlas) Esophageal 7.6886 0.1037 −1.462679 0.910990 −1.682311 −0.362298 Adenocarcinoma (0.7178) (1.0000) (0.4625) (1.0000) (TCGA, PanCancer Atlas) Cholangiocarcin 4.1691 0.1244 −2.037840 0.972100 N/A N/A oma (TCGA, (0.0623) (0.4965) PanCancer Atlas) Brain Lower 4.0473 0.1322 N/A 0.722383 −1.771514 N/A Grade Glioma (0.7051) (0.1147) (TCGA, PanCancer Atlas) Thymoma 4.0322 1.33E−01 N/A 1.982334 0.369115 N/A (TCGA, (0.0712) (1.0000) PanCancer Atlas) Pediatric Acute 5.5309 0.1368 1.437404 −0.805100 1.607586 N/A Lymphoid (0.4518) (1.0000) (0.3238) Leukemia- Phase II (TARGET, 2018) Diffuse Large B- 1.744 0.1866 N/A 1.320613 N/A N/A Cell Lymphoma (0.0933) (TCGA, PanCancer Atlas) Glioblastoma 2.9376 0.2302 N/A 1.428778 −0.716110 N/A Multiforme (0.2296) (0.7109) (TCGA, PanCancer Atlas) Metastatic 4.069 0.254 −1.812613 0.992571 0.3140 N/A Prostate (0.2097) (0.9628) (1.0000) Cancer, SU2C/PCF Dream (Robinson et al., Cell 2015) Acute Myeloid 2.4016 0.301 N/A −1.539142 −0.199532 N/A Leukemia (0.1857) (1.0000) (TCGA, PanCancer Atlas) Testicular Germ 3.3144 0.3456 0.574846 −0.443110 −1.751161 N/A Cell Tumors (1.0000) (1.0000) (0.2398) (TCGA, PanCancer Atlas) Adrenocortical 2.0003 0.3678 N/A 1.346397 0.550103 N/A Carcinoma (0.2673) (0.8734) (TCGA, PanCancer Atlas) Uterine 2.44 0.4862 N/A 0.476071 −0.550292 1.2151 Carcinosarcoma (1.0000) (1.0000) (0.6730) (TCGA, PanCancer Atlas) Pancreatic 3.3833 4.96E−01 −1.195082 0.159442 −0.602558 1.217697 Adenocarcinoma (1.0000) (1.0000) (1.0000) (1.0000) (TCGA, PanCancer Atlas) Prostate 1.3139 0.5184 N/A −0.406579 −1.089948 N/A Adenocarcinoma (1.0000) (0.4136) (MSKCC, Cancer Cell 2010) Prostate 0.028351 0.9859 N/A 0.160404 0.079785 N/A Adenocarcinoma (1.0000) (1.0000) (Fred Hutchinson CRC, Nat Med 2016) 

1-31. (canceled)
 32. A method of preventing, suppressing or treating cancer, the method comprising administering to a patient in need thereof a peptide ligand specific for EphA2, or a pharmaceutically acceptable salt or modified derivative thereof, wherein the peptide ligand comprises a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a non-aromatic molecular scaffold which forms covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, wherein the polypeptide comprises an amino acid sequence selected from (SEQ ID NO: 1) C(HyP)LVNPLCLHP(D-Asp)W(HArg)C; (SEQ ID NO: 2) (β-Ala)-Sar₁₀-A(HArg)DC(HyP)LVNPLCLHP(D- Asp)W(HArg)C;  (SEQ ID NO: 3) ACMNDWWCAMGWKCA;  (SEQ ID NO: 4) ACVPDRRCAYMNVCA;  (SEQ ID NO: 5) ACVVDGRCAYMNVCA;  (SEQ ID NO: 6) ACVVDSRCAYMNVCA; (SEQ ID NO: 7) ACVPDSRCAYMNVCA;  (SEQ ID NO: 8) ACYVGKECAIRNVCA; (SEQ ID NO: 9) ACYVGKECAYMNVCA;  (SEQ ID NO: 10) ARDCPLVNPLCLHPGWTC;  (SEQ ID NO: 11) A(HArg)DCPLVNPLCLHPGWTC;  (SEQ ID NO: 12) CPLVNPLCLHPGWTCLHG;  (SEQ ID NO: 13) CPLVNPLCLHPGWTCL(D-His)G; (SEQ ID NO: 14) CPLVNPLCLHPGWSCRGQ; (SEQ ID NO: 15) CPLVNPLCLHPGWSC(HArg)GQ;  (SEQ ID NO: 16) ACVPDRRCAYMNVC;  (SEQ ID NO: 17) DLRCGGDPRCAYMNVCA;  (SEQ ID NO: 18) SRPCVIDSRCAYMNVCA;  (SEQ ID NO: 19) ESRCSPDARCAYMNVCA;  (SEQ ID NO: 20) HSGCRPDPRCAYMNVCA;  (SEQ ID NO: 21) GSGCKPDSRCAYMNVCA;  (SEQ ID NO: 22) ETVCLPDSRCAYMNVCA;  (SEQ ID NO: 23) GQVCIVDARCAYMNVCA;  (SEQ ID NO: 24) ACVPDRRCAFENVCVDH;  (SEQ ID NO: 25) ACVPDRRCAFMNVCEDR;  (SEQ ID NO: 26) ACVPDRRCAFQDVCDHE;  (SEQ ID NO: 27) ACVPDRRCAFRDVCLTG; (SEQ ID NO: 28) ACQPSNHCAFMNYCA;  (SEQ ID NO: 29) ACSPTPACAVQNLCA;  (SEQ ID NO: 30) ACTSCWAYPDSFCA; (SEQ ID NO: 31) ACTKPTGFCAYPDTICA;  (SEQ ID NO: 32) ACRGEWGYCAYPDTICA;  (SEQ ID NO: 33) ACRNWGMYCAYPDTICA;  (SEQ ID NO: 34) ACPDWGKYCAYPDTICA;  (SEQ ID NO: 35) ACRVYGPYCAYPDTICA;  (SEQ ID NO: 36) ACSSCWAYPDSVCA;  (SEQ ID NO: 37) ACQSCWAYPDTYCA;  (SEQ ID NO: 38) ACGFMGLEPCETFCA;  (SEQ ID NO: 39) ACGFMGLVPCEVHCA;  (SEQ ID NO: 40) ACGFMGLEPCEMVCA;  (SEQ ID NO: 41) ACGFMGLEPCVTYCA;  (SEQ ID NO: 42) ACGFMGLEPCELVCA;  (SEQ ID NO: 43) ACGFMGLVPCNVFCA;  (SEQ ID NO: 44) ACGFMGLEPCELFCA;  (SEQ ID NO: 45) ACGFMGLEPCELFCMPK;  (SEQ ID NO: 46) ACGFMGLEPCELYCA;  (SEQ ID NO: 47) ACGFMGLEPCELYCAHT;  (SEQ ID NO: 48) ACGFMGLEPCEMYCA; (SEQ ID NO: 49) ACGFMGLVPCELYCADN;  (SEQ ID NO: 50) ACPLVNPLCLTSGWKCA;  (SEQ ID NO: 51) ACPMVNPLCLHPGWICA;  (SEQ ID NO: 52) ACPLVNPLCLHPGWICA;  (SEQ ID NO: 53) ACPLVNPLCLHPGWRCA;  (SEQ ID NO: 54) ACPLVNPLCNLPGWTCA;  (SEQ ID NO: 55) ACPLVNPLCLVPGWSCA;  (SEQ ID NO: 56) ACPLVNPLCLLDGWTCA;  (SEQ ID NO: 57) ACPLVNPLCLMPGWGCA;  (SEQ ID NO: 58) ACPLVNPLCMIGNWTCA;  (SEQ ID NO: 59) ACPLVNPLCLMTGWSCA;  (SEQ ID NO: 60) ACPLVNPLCMMGGWKCA;  (SEQ ID NO: 61) ACPLVNPLCLYGSWKCA;  (SEQ ID NO: 62) ACPLVNPLCLHPGWTCA;  (SEQ ID NO: 63) ARDCPLVNPLCLHPGWTCA;  (SEQ ID NO: 64) RPACPLVNPLCLHPGWTCA;  (SEQ ID NO: 65) RPPCPLVNPLCLHPGWTCA;  (SEQ ID NO: 66) KHSCPLVNPLCLHPGWTCA;  (SEQ ID NO: 67) ACPLVNPLCLHPGWTCLHG;  (SEQ ID NO: 68) ACPLVNPLCLHPGWTCL(D-His)G;  (SEQ ID NO: 69) ACPLVNPLCLHPG(2Nal)TCLHG;  (SEQ ID NO: 70) RHDCPLVNPLCLLPGWTCA;  (SEQ ID NO: 71) TPRCPLVNPLCLMPGWTCA; (SEQ ID NO: 72) ACPLVNPLCLAPGWTCA;  (SEQ ID NO: 73) ACPLVNPLCLAPGWTCSRS;  (SEQ ID NO: 74) ACPLVNPLCLEPGWTCA;  (SEQ ID NO: 75) ACPLVNPLCLEPGWTCAKR;  (SEQ ID NO: 76) ACPLVNPLCLHPGWSCA;  (SEQ ID NO: 77) ACPLVNPLCLHPGWSCRGQ;  (SEQ ID NO: 78) ACPLVNPLCLHPGWSC(HArg)GQ;  (SEQ ID NO: 79) ACPLVNPLCLHPG(2Nal)SCRGQ;  (SEQ ID NO: 80) ACPLVNPLCLTPGWTCTNT;  (SEQ ID NO: 81) ACPMVNPLCLHPGWKCA;  (SEQ ID NO: 82) ACPMVNPLCLTPGWICA;  (SEQ ID NO: 83) ACPMVNPLCLHPGWTCA;  (SEQ ID NO: 84) H(D-Asp)VT-C(Aib)(1Nal)G(Aib)F(1Nal)CP(tBuGly) N(HArg)P(D-Asp)C;  (SEQ ID NO: 85) A(HArg)DC(HyP)(Hse(Me))VNPLCLHP(D-Asp)W(HArg)C;  (SEQ ID NO: 86) A(HArg)DC(HyP)(Hse(Me))VNPLCLHP(D-Asp)WTC;  (SEQ ID NO: 87) A(HArg)DC(HyP)LVNPLCLHP(D-Ala)WTC;  (SEQ ID NO: 88) A(HArg)DCPLVNPLCLHP(D-Ala)WTC;  (SEQ ID NO: 89) ARDC(HyP)LVNPLCLHPGWTC; (SEQ ID NO: 90) ARDCPLVNPLCL(D-3,3-DPA)PGWTC;  (SEQ ID NO: 91) ARDCPLVNPLCLHPGWTCLH;  (SEQ ID NO: 92) A(HArg)DCPLVNPLCLHP(D-Cya)WTC;  (SEQ ID NO: 93) A(D-Arg)DC(HyP)LVNPLCL(D-3,3-DPA)P(D-Asp)W(HArg)C; (SEQ ID NO: 94) ACPWGPAWCPVNRPGCA;  (SEQ ID NO: 95) ACPWGPFWCPVNRPGCA;  (SEQ ID NO: 96) ADVTCPWGPFWCPVNRPGCA; and (SEQ ID NO: 97) CPLVNPLCLHPGWTC;

wherein HyP is hydroxyproline, HArg is homoarginine, Sar is sarcosine, HArg is homoarginine, D-Asp is D-aspartic acid, 2Nal is 2-naphthylalanine, 1Nal is 1-naphthylalanine, Aib is 2-aminoisobutyric acid, tBuGly is tert-leucine, hSerMe is homoserine(methyl), D-Ala is D-alanine, D-3,3-DPA is 3,3-diphenyl-D-alanine, D-Cya is D-cysteic acid, D-Arg is D-arginine, HPhe is homophenylalanine, and D-His is D-histidine.
 33. The method of claim 32, wherein the polypeptide comprises an amino acid sequence selected from ACMNDWWCAMGWKCA-Sar₆-K(FI) ((SEQ ID NO: 3)-Sar₆-K(FI)); ACVPDRRCAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 4)-Sar₆-K(FI)); ACVVDGRCAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 5)-Sar₆-K(FI)); ACVVDSRCAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 6)-Sar₆-K(FI)); ACVPDSRCAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 7)-Sar₆-K(FI)); ACYVGKECAIRNVCA-Sar₆-K(FI) ((SEQ ID NO: 8)-Sar₆-K(FI)); ACYVGKECAYMNVCA-Sar₆-K(FI) ((SEQ ID NO: 9)-Sar₆-K(FI)); FI-G-Sar₅-ACYVGKECAYMNVCA (FI-G-Sar₅-(SEQ ID NO: 9)); FI-(β-Ala)-Sar₁₀-ARDCPLVNPLCLHPGWTC (FI-(β-Ala)-Sar₁₀-(SEQ ID NO: 10)); FI-(β-Ala)-Sar₁₀-A(HArg)DCPLVNPLCLHPGWTC (FI-(β-Ala)-Sar₁₀-(SEQ ID NO: 11); Ac-CPLVNPLCLHPGWTCLHG-Sar₆-(D-K[FI]) (Ac-(SEQ ID NO: 12)-Sar₆-(D-K[FI])); Ac-CPLVNPLCLHPGWTCL(D-His)G-Sar₆-(D-K[FI]) (Ac-SEQ ID NO: 13)-Sar₆-(D-K[FI])); Ac-CPLVNPLCLHPGWSCRGQ-Sar₆-(D-K[FI]) (Ac-(SEQ ID NO: 14)- Sar₆-(D-K[FI])); Ac-CPLVNPLCLHPGWSC(HArg)GQ-Sar₆-(D-K[FI]) (Ac-(SEQ ID NO: 15)- Sar₆-(D-K[FI])); (β-Ala)-Sar₁₀-ACVPDRRCAYMNVC ((β-Ala)-Sar₁₀-(SEQ ID NO: 16)); (β-Ala)-Sar₁₀-A(HArg)DCPLVNPLCLHPGWTC ((β-Ala)-Sar₁₀-(SEQ ID NO: 11); Ac-ARDCPLVNPLCLHPGWTCA-Sar₆-(D-K) (Ac-(SEQ ID NO: 63)-Sar₆-(D-K)); Ac-A(HArg)DCPLVNPLCLHPGWTCA- Sar₆-(D-K) (Ac-(SEQ ID NO: 11)-A-Sar₆-(D-K)); Ac-CPLVNPLCLHPGWTCLHG (Ac-(SEQ ID NO: 12)); (β-Ala)-Sar₁₀-ACPLVNPLCLHPGWTCLHG ((β-Ala)-Sar₁₀-(SEQ ID NO: 67)); (β-Ala)-Sar₁₀-ACPLVNPLCLHPGWTCL(D-His)G ((β-Ala)-Sar₁₀-(SEQ ID NO: 68)); Ac-CPLVNPLCLHPGWTCLHG-Sar₆-(D-K) (Ac-(SEQ ID NO: 12)-Sar₆-(D-K)); Ac-CPLVNPLCLHPGWTCL(D-His)G-Sar₆-(D-K) (Ac-(SEQ ID NO: 13)- Sar₆-(D-K)); Ac-CPLVNPLCLHPGWSCRGQ (Ac-(SEQ ID NO: 14)); (β-Ala)-Sar₁₀-ACPLVNPLCLHPGWSCRGQ ((β-Ala)-Sar₁₀-(SEQ ID NO: 77); (β-Ala)-Sar₁₀-ACPLVNPLCLHPGWSC(HArg)GQ ((β-Ala)-Sar₁₀-(SEQ ID NO: 78)); Ac-CPLVNPLCLHPGWSCRGQ-Sar₆-(D-K) (Ac-(SEQ ID NO: 14)-Sar₆-(D-K)); Ac-CPLVNPLCLHPGWSC(HArg)GQ-Sar₆-(D-K) (Ac-(SEQ ID NO: 15) -Sar₆-(D-K)); (β-Ala)-Sar₁₀-H(D-Asp)VT-C(Aib)(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)P(D-Asp)C ((β-Ala)- Sar₁₀-(SEQ ID NO: 84)); (β-Ala)-Sar₁₀-(SEQ ID NO: 10); (β-Ala)-Sar₁₀-(SEQ ID NO: 11)-CONH₂; (β-Ala)-Sar₅-(SEQ ID NO: 11); Ac-(SEQ ID NO: 12)-Sar₆-(D-K); Ac-(SEQ ID NO: 14)-Sar₆-(D-K); Ac-(SEQ ID NO: 12)-Sar₆-(D-K[Ac]); (β-Ala)-Sar₁₀-(SEQ ID NO: 2)-CONH₂; (β-Ala)-Sar₁₀-(SEQ ID NO: 87); (β-Ala)-Sar₁₀-(SEQ ID NO: 88); (β-Ala)-Sar₁₀-(SEQ ID NO: 86); (β-Ala)-Sar₁₀-(SEQ ID NO: 85); (β-Ala)-Sar₁₀-(SEQ ID NO: 91); (β-Ala)-Sar₁₀-(SEQ ID NO: 90); (β-Ala)-Sar₁₀-(SEQ ID NO: 89); (β-Ala)-Sar₁₀-(SEQ ID NO: 93); (β-AlaSO₃H)-Sar₁₀-(SEQ ID NO: 11); (β-AlaSO₃H)-Sar₅-(SEQ ID NO: 11); and (β-AlaSO₃H)-Sar₄-(Cya)-Sar₄-(Cya)-(SEQ ID NO: 92).


34. The method of claim 32, wherein the polypeptide comprises the amino acid sequence (SEQ ID NO: 1) C(HyP)LVNPLCLHP(D-Asp)W(HArg)C.


35. The method of claim 32, wherein the polypeptide comprises the amino acid sequence (β-Ala)-Sar₁₀-A(HArg)DC(HyP)LVNPLCLHP(D-Asp) W(HArg)C (SEQ ID NO: 2); or (SEQ ID NO: 2)-CONH₂.


36. The method of claim 32, wherein the polypeptide comprises the amino acid sequence selected from A(HArg)DCPLVNPLCLHPGWTC (SEQ ID NO: 11); FI-(β-Ala)-Sar₁₀-(SEQ ID NO: 11); (β-Ala)-Sar₁₀-(SEQ ID NO: 11); Ac-(SEQ ID NO: 11)-A-Sar₆-(D-K); (β-Ala)-Sar₁₀-(SEQ ID NO: 11)-CONH₂; (β-Ala)-Sar₅-(SEQ ID NO: 11); (β-AlaSO₃H)-Sar₁₀-(SEQ ID NO: 11); and (β-AlaSO₃H)-Sar₅-(SEQ ID NO: 11).


37. The method of claim 32, wherein the polypeptide comprises the amino acid sequence (SEQ ID NO: 97) CPLVNPLCLHPGWTC


38. The method of claim 32, wherein the molecular scaffold is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).
 39. The method of claim 32, wherein the peptide ligand is selected from

or a pharmaceutically acceptable salt thereof.
 40. The method of claim 32, wherein the pharmaceutically acceptable salt is selected from a free acid or a sodium, potassium, calcium, or ammonium salt.
 41. The method of claim 32, wherein the EphA2 is human EphA2.
 42. The method of claim 32, wherein the peptide ligand is conjugated to one or more effector and/or functional group forming a drug conjugate.
 43. The method of claim 42, wherein said effector and/or functional group is a cytotoxic agent selected from DM1 and MMAE.
 44. The method of claim 43, wherein the drug conjugate comprises a linker between said peptide ligand and said cytotoxic agent.
 45. The method of claim 44, wherein said cytotoxic agent is MMAE and the linker is selected from: -Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-, -Ala-Ala-Asn-, D-Ala-Phe-Lys-, and -Glu-Pro-Cit-Gly-hPhe-Tyr-Leu- (SEQ ID NO: 98).
 46. The method of claim 44, wherein said cytotoxic agent is MMAE and the linker is -Val-Cit-.
 47. The method of claim 44, wherein said cytotoxic agent is DM1 and the linker is selected from: —S—S—, —SS(SO₃H)—, —SS-(Me)-, -(Me)-SS-(Me)-, —SS-(Me₂)-, and —SS-(Me)-SO₃H—.
 48. The method of claim 42, wherein the drug conjugate is selected from

and or a pharmaceutically acceptable salt thereof, wherein BCY6099 has a structure

wherein BCY6014 has a structure

wherein BCY6104 has a structure

wherein BCY6018 has a structure

wherein BCY 6017 as a structure

wherein BCY6019 has a structure

wherein BCY 6009 has a structure

wherein BCY6152 has a structure

wherein BCY6153 has a structure

wherein BCY6138 has a structure

and wherein BCY6137 has a structure


49. The method of claim 32, wherein the cancer is selected from prostate cancer, lung cancer (such as non-small cell lung carcinomas (NSCLC)), breast cancer (such as triple negative breast cancer), gastric cancer, ovarian cancer, oesophageal cancer, multiple myeloma, and fibrosarcoma.
 50. The method of claim 49, wherein the lung cancer is non-small cell lung carcinomas (NSCLC)), and the breast cancer is triple negative breast cancer.
 51. The method of claim 32, wherein the patient has an increased copy number variation (CNV) of EphA2. 